Nucleic acid mediated electron transfer

ABSTRACT

The present invention provides for the selective covalent modification of nucleic acids with redox active moieties such as transition metal complexes. Electron donor and electron acceptor moieties are covalently bound to the ribose-phosphate backbone of a nucleic acid at predetermined positions. The resulting complexes represent a series of new derivatives that are bimolecular templates capable of transferring electrons over very large distances at extremely fast rates. These complexes possess unique structural features which enable the use of an entirely new class of bioconductors and photoactive probes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuing application of U.S. Ser. No.09/306,749, filed May 7, 1999, which is a continuing application of Ser.No. 08/873,598, filed Jun. 12, 1997, U.S. Pat. No. 5,952,172, which is acontinuing application of U.S. Ser. No. 08/660,534, filed Jun. 7, 1996,U.S. Pat. No. 5,770,369, which is a continuing application of U.S. Ser.No. 08/475,051, filed Jun. 7, 1995, U.S. Pat. No. 5,824,473, which is acontinuing application of U.S. Ser. No. 08/166,036, filed Dec. 10, 1993,U.S. Pat. No. 5,591,578.

FIELD OF THE INVENTION

The present invention is directed to electron transfer via nucleicacids. More particularly, the invention is directed to improvements inthe site-selective modification of nucleic acids with electron transfermoieties.

BACKGROUND OF THE INVENTION

The detection of specific nucleic acid sequences is an important toolfor diagnostic medicine and molecular biology research. Gene probeassays currently play roles in identifying infectious organisms such asbacteria and viruses, in probing the expression of normal genes andidentifying mutant genes such as oncogenes, in typing tissue forcompatibility preceding tissue transplantation, in matching tissue orblood samples for forensic medicine, and for exploring homology amonggenes from different species.

Ideally, a gene probe assay should be sensitive, specific and easilyautomatable (for a review, see Nickerson, Current Opinion inBiotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. lowdetection limits) has been greatly alleviated by the development of thepolymerase chain reaction (PCR) and other amplification technologieswhich allow researchers to amplify exponentially a specific nucleic acidsequence before analysis (for a review, see Abramson et al., CurrentOpinion in Biotechnology, 4:41-47 (1993)).

Specificity, in contrast, remains a problem in many currently availablegene probe assays. The extent of molecular complementarity between probeand target defines the specificity of the interaction. Variations in theconcentrations of probes, of targets and of salts in the hybridizationmedium, in the reaction temperature, and in the length of the probe mayalter or influence the specificity of the probe/target interaction.

It may be possible under some limited circumstances to distinguishtargets with perfect complementarity from targets with mismatches,although this is generally very difficult using traditional technology,since small variations in the reaction conditions will alter thehybridization. New experimental techniques for mismatch detection withstandard probes include DNA ligation assays where single pointmismatches prevent ligation and probe digestion assays in whichmismatches create sites for probe cleavage.

Finally, the automation of gene probe assays remains an area in whichcurrent technologies are lacking. Such assays generally rely on thehybridization of a labeled probe to a target sequence followed by theseparation of the unhybridized free probe. This separation is generallyachieved by gel electrophoresis or solid phase capture and washing ofthe target DNA, and is generally quite difficult to automate easily.

The time consuming nature of these separation steps has led to twodistinct avenues of development. One involves the development ofhigh-speed, high-throughput automatable electrophoretic and otherseparation techniques. The other involves the development ofnon-separation homogeneous gene probe assays.

For example, Gen-Probe Inc., (San Diego, Calif.) has developed ahomogeneous protection assay in which hybridized probes are protectedfrom base hydrolysis, and thus are capable of subsequentchemiluminescence. (Okwumabua et al. Res. Microbiol. 143:183 (1992)).Unfortunately, the reliance of this approach on a chemiluminescentsubstrate known for high background photon emission suggests this assaywill not have high specificity. EPO application number 86116652.8describes an attempt to use non-radiative energy transfer from a donorprobe to an acceptor probe as a homogeneous detection scheme. However,the fluorescence energy transfer is greatly influenced by both probetopology and topography, and the DNA target itself is capable ofsignificant energy quenching, resulting in considerable variability.Therefore there is a need for DNA probes which are specific, capable ofdetecting target mismatches, and capable of being incorporated into anautomated system for sequence identification.

As outlined above, molecular biology relies quite heavily on modified orlabeled oligonucleotides for traditional gene probe assays(Oligonucleotide Synthesis: A Practical Approach. Gait et al., Ed., IRLPress: Oxford, UK, 1984; Oligonucleotides and Analogues: A PracticalApproach. Ed. F. Eckstein, Oxford University Press, 1991). As a result,several techniques currently exist for the synthesis of tailored nucleicacid molecules. Since nucleic acids do not naturally contain functionalgroups to which molecules of interest may easily be attached covalently,methods have been developed which allow chemical modification at eitherof the terminal phosphates or at the heterocyclic bases (Dreyer et al.Proc. Natl. Acad. Sci. USA, 1985, 82:968).

For example, analogues of the common deoxyribo- and ribonucleosideswhich contain amino groups at the 2′ or 3′ position of the sugar can bemade using established chemical techniques. (See Imazawa et al., J. Org.Chem., 1979, 44:2039; Imazawa et al., J. Org. Chem. 43(15):3044 (1978);Verheyden et al., J. Org. Chem. 36(2):250 (1971); Hobbs et al., J. Org.Chem. 42(4):714 (1977)). In addition, oligonucleotides may besynthesized with 2′-5′ or 3′-5′ phosphoamide linkages (Beaucage et al.,Tetrahedron 49(10):1925 (1992); Letsinger, J. Org. Chem., 35:3800(1970); Sawai, Chem. Left. 805 (1984); Oligonucleotides ;and Analogues:A Practical Approach, F. Eckstein, Ed. Oxford University Press (1991)).

The modification of nucleic acids has been done for two general reasons:to create nonradioactive DNA markers to serve as probes, and to usechemically modified DNA to obtain site-specific cleavage.

To this end, DNA may be labeled to serve as a probe by altering anucleotide which then serves as a replacement analogue in the nicktranslational resynthesis of double stranded DNA. The chemically alterednucleotides may then provide reactive sites for the attachment ofimmunological or other labels such as biotin. (Gilliam et al., Anal.Biochem. 157:199 (1986)). Another example uses ruthenium derivativeswhich intercalate into DNA to produce photoluminescence under definedconditions. (Friedman et al., J. Am. Chem. Soc. 112:4960 (1990)).

In the second category, there are a number of examples of compoundscovalently linked to DNA which subsequently cause DNA chain cleavage.For example 1,10-phenanthroline has been coupled to single-strandedoligothymidylate via a linker which results in the cleavage of poly-dAoligonucleotides in the presence of Cu²⁺ and 3-mercaptopropionic acid(Francois et al., Biochemistry 27:2272 (1988)). Similar experiments havebeen done for EDTA¹-Fe(II) (both for double stranded DNA (Boutorin etal., FEBS Lett. 172:43-46 (1986)) and triplex DNA (Strobel et al.,Science 249:73 (1990)), porphyrin-Fe(III) (Le Doan et al., Biochemistry25:6736-6739 (1986)), and 1,10-phenanthronine-Cu(I) (Chen et al., Proc.Natl. Acad. Sci USA, 83:7147 (1985)), which all result in DNA chaincleavage in the presence of a reducing agent in aerated solutions. Asimilar example using porphyrins resulted in DNA strand cleavage, andbase oxidation or cross-linking of the DNA under very specificconditions (Le Doan et al., Nucleic Acids Res. 15:8643 (1987)).

Other work has focused on chemical modification of heterocyclic bases.For example, the attachment of an inorganic coordination complex,Fe-EDTA, to a modified internal base resulted in cleavage of the DNAafter hybridization in the presence of dioxygen (Dreyer et al., Proc.Natl. Acad. Sci. USA 82:968 (1985)). A ruthenium compound has beencoupled successfully to an internal base in a DNA octomer, withretention of both the DNA hybridization capabilities as well as thespectroscopic properties of the ruthenium label (Telser et al., J. Am.Chem. Soc. 111:7221 (1989)). Other experiments have successfully addedtwo separate spectroscopic labels to a single double-stranded DNAmolecule (Telser et al., J. Am. Chem. Soc. 111:7226 (1989)).

The study of electron transfer reactions in proteins and DNA has alsobeen explored in pursuit of systems which are capable of long distanceelectron transfer.

To this end, intramolecular electron transfer in protein—proteincomplexes, such as those found in photosynthetic proteins and proteinsin the respiration pathway, has been shown to take place overappreciable distances in protein interiors at biologically significantrates (see Bowler et al., Progress in Inorganic Chemistry: BioinorganicChemistry, Vol. 38, Ed. Stephen J. Lippard (1990). In addition, theselective modification of metalloenzymes with transition metals has beenaccomplished and techniques to monitor electron transfer in thesesystems developed. For example, electron transfer proteins such ascytochrome c have been modified with ruthenium through attachment atseveral histidines and the rate of electron transfer from the heme Fe²⁺to the bound Ru³⁺ measured. The results suggest that electron transfer“tunnel” pathways may exist. (Baum, Chemical & Engineering News, Feb.22, 1993, pages 2023; see also Chang et al., J. Am. Chem. Soc. 113:7056(1991)). In related work, the normal protein insulation, which protectsthe redox centers of an enzyme or protein from nondiscriminatoryreactions with the exterior solvent, was “wired” to transform thesesystems from electrical insulators into electrical conductors (Heller,Acc. Chem. Res. 23:128 (1990)).

There are a few reports of photoinduced electron transfer in a DNAmatrix. In these systems, the electron donors and acceptors are notcovalently attached to the DNA, but randomly associated with the DNA,thus rendering the explicit elucidation and control of thedonor-acceptor system difficult. For example, the intense fluorescenceof certain quaternary diazoaromatic salts is quenched upon intercalationinto DNA or upon exposure to individual mononucleotides, thus exhibitingelectron donor processes within the DNA itself. (Brun et al., J. Am.Chem. Soc. 113:8153 (1991)).

Another example of the difficulty of determining the electron transfermechanism is found in work done with some photoexcitable rutheniumcompounds. Early work suggested that certain ruthenium compounds eitherrandomly intercalate into the nucleotide bases, or bind to the helixsurface. (Purugganan et al., Science 241:1645 (1988)). A recentreference indicates that certain ruthenium compounds do not intercalateinto the DNA (Satyanarayana et al., Biochemistry 31(39):9319 (1992));rather, they bind non-covalently to the surface of the DNA helix.

In these early experiments, various electron acceptor compounds, such ascobalt, chromium or rhodium compounds were added to certainDNA-associated ruthenium electron donor compounds. (Puragganan et al.,Science 241:1645 (1988); Orellana et al., Photochem. Photobiol. 499:54(1991); Brun et al., J. Am. Chem. Soc. 113:8153 (1991); Davis,Chem.-Biol. Interactions 62:45 (1987); Tomalia et al., Acc. Chem. Res.,24:332 (1991)). Upon addition of these various electron acceptorcompounds, which randomly bind non-covalently to the helix, quenching ofthe photoexcited state through electron transfer was detected. The rateof quenching was dependent on both the individual electron donor andacceptor as well as their concentrations, thus revealing the process asbimolecular.

In one set of experiments, the authors postulate that the more mobilesurface bound donor promotes electron transfer with greater efficiencythan the intercalated species, and suggest that the sugar-phosphatebackbone of DNA, and possibly the solvent medium surrounding the DNA,play a significant role in the electron transport. (Purugganan et al.,Science 241:1645 (1988)). In other work, the authors stress thedependence of the rate on the mobility of the donor and acceptor andtheir local concentrations, and assign the role of the DNA to beprimarily to facilitate an increase in local concentration of the donorand acceptor species on the helix. (Orellana et al., supra).

In another experiment, an electron donor was reportedly randomlyintercalated into the stack of bases of DNA, while the acceptor wasrandomly associated with the surface of the DNA. The rate of electrontransfer quenching indicated a close contact of the donor and theacceptor, and the system also exhibits enhancement of the rate ofelectron transfer with the addition of salt to the medium. (Fromherz etal., J. Am. Chem. Soc. 103:5361 (1986)).

In all of these experiments, the rate of electron transfer fornon-covalently bound donors and acceptors is several orders of magnitudeless than is seen in free solution.

An important stimulus for the development of long distance electrontransfer systems is the creation of synthetic light harvesting systems.Work to date suggests that an artificial light harvesting systemcontains an energy transfer complex, an energy migration complex, anelectron transfer complex and an electron migration complex (for atopical review of this area, see Chemical & Engineering News, Mar. 15,1993, pages 38-48). Two types of molecules have been tried: a) longorganic molecules, such as hydrocarbons with covalently attachedelectron transfer species, or DNA, with intercalated, partiallyintercalated or helix associated electron transfer species, and b)synthetic polymers.

The long organic molecules, while quite rigid, are influenced by anumber of factors, which makes development difficult. These factorsinclude the polarity and composition of the solvent, the orientation ofthe donor and acceptor groups, and the chemical character of either thecovalent linkage or the association of the electron transfer species tothe molecule.

The creation of acceptable polymer electron transfer systems has beendifficult because the available polymers are too flexible, such thatseveral modes of transfer occur. Polymers that are sufficiently rigidoften significantly interfere with the electron transfer mechanism orare quite difficult to synthesize.

Thus the development of an electron transfer system which issufficiently rigid, has covalently attached electron transfer species atdefined intervals, is easy to synthesize and does not appreciablyinterfere with the electron transfer mechanism would be useful in thedevelopment of artificial light harvesting systems.

In conclusion, the random distribution and mobility of the electrondonor and acceptor pairs, coupled with potential short distances betweenthe donor and acceptor, the loose and presumably reversible associationof the donors and acceptors, the reported dependence on solvent andbroad putative electron pathways, and the disruption of the DNAstructure of intercalated compounds rendering normal base pairingimpossible all serve as pronounced limitations of long range electrontransfer in a DNA matrix. Therefore, a method for the production ofrigid, covalent attachment of electron donors and acceptors to provideminimal perturbations of the nucleic acid structure and retention of itsability to base pair normally, is desirable. The present inventionserves to provide such a system, which allows the development of novelbioconductors and diagnostic probes.

SUMMARY OF THE INVENTION

The present invention provides for the modification of nucleic acids atspecific sites with redox active moieties such as transition metalcomplexes. An electron donor and/or electron acceptor moiety arecovalently bound at predetermined positions. The resulting complexesrepresent a series of new derivatives that are biomolecular templatescapable of transferring electrons over very large distances at extremelyfast rates. These complexes possess unique structural features whichenable the use of an entirely new class of bioconductors and diagnosticprobes.

Accordingly, it is an object of the invention to provide nucleic acidswith electron transfer species covalently attached to a terminal base ofthe nucleic acid. It is a further object to provide nucleic acids withcovalently attached organic electron transfer species, and modifiednucleic acids attached to control pore glass.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1H illustrates all the possible orientations of electrondonor (EDM) and electron acceptor (EAM) moieties on a single strandednucleic acid.

FIGS. 2A-1 to 2A-9 and 2B-1 to 2B-9 illustrate the possible orientationsof electron transfer moieties EDM and EAM on two adjacent singlestranded nucleic acids. These orientations also apply when the twoprobes are separated by an intervening sequence.

FIG. 3 illustrates a series of amino-modified nucleoside precursorsprior to incorporation into an oligonucleotide.

FIGS. 4A and 4B depict the structure of electron transfer moieties. FIG.4A depicts the general formula of a representative class of electrondonors and acceptors. FIG. 4B depicts a specific example of a rutheniumelectron transfer moiety using bisbipyridine and imidazole as theligands.

FIG. 5 is a schematic showing transition metals bound to theribose-phosphate backbone in a variety of positions. M is a transitionmetal. M₁ is bound via an amine on the 2′ carbon of the ribose; anelectron must travel through 4 σ bonds to enter the pi-orbitals (the“pi-way”) of the stacked bases. M₂ and M₃ are bound via aphosphoramide-type linkages, and electrons must travel through 7 σ bondsto enter the pi-way, respectively. M₄ is bound via an amine on the 3′carbon of the ribose, and an electron traverses through 5 σ bonds.

FIGS. 6A, 6B and 6C depict the attachment of a 2′-amino-modifiednucleoside to control pore glass (CPG) and the formation of a singlestranded nucleic acid with elongation and attachment of transition metalcomplexes as the exemplified electron transfer species. The experimentalconditions are outlined in Example 9. FIG. 6A depicts the formation of2′-amino-2′-deoxyuridine derivatized to control pore glass (CPG).2′-amino modified uridine is depicted, although any base may be used. Asis known in the art, phosphoramidite nucleosides are added to thederivatized nucleoside, after removal of the DMT protecting group, asgenerally depicted in FIG. 6B, using the UCTCCTACAC sequence as anexample. The addition of a 5′ terminal phosphoramidite2-amino-deoxyuridine, with a DMT protecting group, results in a singlestranded nucleic acid containing a 3′ and 5′2′-amino modifiednucleoside. FIG. 6C depicts the addition of the electron transferspecies, exemplified by two ruthenium transition metal complexes,im(bpy)₂Ru and Ru(II)(NH₃)₄py.

FIG. 7 depicts the addition of electron transfer moieties, exemplifiedby a transition metal complex, to the C-terminus of PNA. FIG. 9 attaches4-aminomethylpyridine to the carboxy terminus, to form a ligand whichmay bind the metal at the nitrogen of the pyridine ring.

FIGS. 8A and 8B depicts attachment of the amino-modified nucleic acidsof the invention to electrodes. (A) depicts the attachment to glassycarbon electrodes. R is the oligonucleotide, and GCE is a glassy carbonelectrode. (B) depicts the attachment of the amino-modified nucleicacids of the invention to oxidized surfaces using silane reactions.

DETAILED DESCRIPTION

Unless otherwise stated, the term “nucleic acid” or “oligonucleotide” orgrammatical equivalents herein means at least two nucleotides covalentlylinked together. A nucleic acid of the present invention will generallycontain phosphodiester bonds, although in some cases, as outlined below,a nucleic acid analogs are included that may have alternate backbones,comprising, for example, phosphoramide (Beaucage et al., Tetrahedron49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem.35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977);Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem.Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988);and Pauwels et al. Chemica Scripta 26:141 91986)), phosphorothioate,phosphorodithioate, O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature380:207 (1996), all of which are incorporated by reference). Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of electron transfer moieties, or to increase the stabilityand half-life of such molecules in physiological environments.

Particularly preferred are peptide nucleic acids (PNA). This backbone issubstantially non-ionic under neutral conditions, in contrast to thehighly charged phosphodiester backbone of naturally occurring nucleicacids. This results in two advantages. First, this backbone exhibitsimproved hybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic backbone of PNA, the drop is closer to 7-9° C. Thisallows for better detection of mismatches. Similarly, due to theirnon-ionic nature, hybridization of the bases attached to these backbonesis relatively insensitive to salt concentration. This is particularlyadvantageous in the systems of the present invention, as a reduced salthybridization solution has a lower Faradaic current than a physiologicalsalt solution (in the range of 150 mM).

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathanine andhypoxathanine, etc. In some instances, e.g. in the case of an“intervening nucleic acid”, the term nucleic acid refers to one or morenucleosides. As used herein, the term “nucleoside” includes nucleotides.

The terms “electron donor moiety”, “electron acceptor moiety”, and“electron transfer moieties” or grammatical equivalents herein refers tomolecules capable of electron transfer under certain conditions. It isto be understood that electron donor and acceptor capabilities arerelative; that is, a molecule which can lose an electron under certainexperimental conditions will be able to accept an electron underdifferent experimental conditions. It is to be understood that thenumber of possible electron donor moieties and electron acceptormoieties is very large, and that one skilled in the art of electrontransfer compounds will be able to utilize a number of compounds in thepresent invention. Preferred electron transfer moieties include, but arenot limited to, transition metal complexes, organic electron transfermoieties, and electrodes.

In a preferred embodiment, the electron transfer moieties are transitionmetal complexes. Transition metals are those whose atoms have anincomplete d shell of electrons. Suitable transition metals for use inthe invention include, but are not limited to, cadmium (Cd), magnesium(Mg), copper (Cu), cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe),ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt),scandium (Sc), titanium (Ti), Vanadium (V), chromium (Cr), manganese(Mn), nickel (Ni), Molybdenum (Mo), technetium (Tc), tungsten (W), andiridium (Ir). That is, the first series of transition metal, theplatinum metals (Ru, Rh, Pd, Os, Ir and Pt), along with Re, W, Mo andTc, are preferred. Particularly preferred are ruthenium, rhenium,osmium, platinium and iron.

The transition metals are complexed with a variety of ligands to formsuitable transition metal complexes, as is well known in the art.Suitable ligands include, but are not limited to, —NH₂; pyridine;pyrazine; isonicotinamide; imidazole; bipyridine and substitutedderivative of bipyridine; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline; dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine; 1,4,5,8-tetraazaphenanthrene(abbreviated tap); 1,4,8,11-tetra-azacyclotetradecane; diaminopyridine(abbreviated damp); porphyrins and substituted derivatives of theporphyrin family. A general formula that is representative of a class ofdonors and acceptors that may be employed is shown in FIG. 4A. Thegroups R¹, R², R³, R⁴, and R⁵ may be any coordinating ligand that iscapable of covalently binding to the chosen metal and may include any ofthe above ligands. The structure of a ruthenium electron transferspecies using bisbipyridine and imidazole as the ligands is shown inFIG. 4B. Specific examples of useful electron transfer complexesinclude, but are not limited to, those shown in Table 1.

TABLE 1 Donors Acceptors Ru(bpy)₂im-NH₂-U Ru(NH₃)₅-NH₂-URu(bpy)₂im-NH₂-U Ru(NH₃)₄py-NH₂-U Ru(bpy)₂im-NH₂-U Ru(NH₃)₄im-NH₂-Utrans-Ru(cyclam)py Where: Ru = ruthenium bpy = bisbipyridine im =imidazole py = pyridine cyclam = 1,4,8,11-tetra-azacyclotetradecane

Where:

Ru=ruthenium

bpy=bisbipyridine

im=imidazole

py=pyridine

cyclam=1,4,8,11-tetra-azacyclotetradecane

Other suitable moieties include bis(phenanthroline)

(dipyridophenazine)Ru(II) (abbreviated [Ru(phen)₂dppz]⁺²);

bis(9,10-phenthrenequinone diimine)(phenanthroline)Rh(III), abbreviated[Rh(phi)₂phen]⁺³;

tris(phenanthroline)Ru(II) (abbreviated [Ru(o-phen)₃]⁺²), Co(phen)₃ ⁺³,Co(bpy)₃ ⁺³; Rh(phen)₃ ⁺³;

Cr(phen )₃ ⁺³; Ru(bpy)₂(dppz)⁺²; and

Ru(bpy)₃ ⁺².

In addition to transition metal complexes, other organic electron donorsand acceptors may be covalently attached to the nucleic acid for use inthe invention. These organic molecules include, but are not limited to,riboflavin, xanthene dyes, azine dyes, acridine orange,N,N′-dimethyl-2,7-diazapyrenium dichloride (DAP²⁺), methylviologen,ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def:6,5,10-d′e′f )diisoquinoline dichloride(ADS IQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, and subsitituted derivatives of these compounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

In a particularly preferred embodiment, an electron transfer moietycomprises an solid support such as an electrode to which the nucleicacid is attached, covalently or otherwise. That is, the electrode servesas either the electron donor or acceptor, as is more fully describedbelow. The techniques used in this embodiment are analogous to thewiring of proteins to an electrode except that the nucleic acids of thepresent invention are used rather than a redox protein (see for exampleGregg et al., J. Phys. Chem. 95:5970 (1991); Heller et al., Sensors andActuators R., 13-14:180 (1993); and Pishko et al., Anal. Chem., 63:2268(1991)).

Electrode attachment is utilized in initiating electron transfer via anapplied potential and for electronic methods of electron transfermonitoring.

In a preferred embodiment, electron transport between the electrode andthe nucleic acid can be indirect, utilizing electron transport mediatorswhich are free in solution or imbedded in a gel or polymer to provide atype of electronic coupling between the electrode and the nucleic acids.In a preferred embodiment, the electron transfer moiety-modified nucleicacids of the invention are attached via such a matrix. Matrix attachmenthas several advantages for use in a nucleic acid gene sensor. Because ofthe 3-dimensional nature of the polymer, large numbers of modifiednucleic acid probes can be attached to a small surface area ofelectrode. Using a highly porous “hydrogel,” rates of nucleic acidhybridization can be quite high, nearly matching that of nucleic acid insolution.

For example, polymers with covalently attached redox molecules behave ashighly effective electron transfer mediators. Siloxane and ethyleneoxide polymers, modified with ferrocene molecules, demonstrated electrontransfer between enzymes and an electrode; for example, flexiblesiloxane and ethylene oxide polymers covalently attached to ferrocene orOs(bpy)₂ have been shown to be highly effective redox polymers formediating electron transfer from several enzymes to an electrode. (seeBoguslavsky et al., Solid State Ionics, V. 60, p. 189, (1993)).Similarly, a redox-conducting epoxy cement has been prepared (see Hellaret al., J. Phys. Chem., 95:5970 (1991)). Cross linked redox gels foramperometric biosensors applications have also been prepared withglucose oxidase electrically connected to electrodes so that electronswere shown to flow from the enzyme, through the polymer and to theelectrode (see Hellar, A., et. al., Anal. Chem., 62, 258, (1990)).

In this embodiment, it is preferred that a redox polymer such as apoly-(vinylpyridine) complex of Os(bby)₂Cl be cross-linked with anepoxide such as diepoxide to form a redox-conducting epoxide cementwhich is capable of strongly binding to electrodes made of conductivematerial such as gold, vitreous carbon, graphite, and other conductivematerials. This strong attachment is included in the definition of“covalently attached” for the purposes of this embodiment. The epoxidecross-linking polymer is then reacted with, for example, an exposedamine, such as the amine of an amino-modified nucleic acid describedabove, covalently attaching the nucleic acid to the complex, forming a“redox hydrogel” on the surface of the electrode.

In an analogous fashion, chemically modified DNA can be substituted forthe redox enzyme or mediator with the result of electron transferprocesses being observed from a transition metal-modified DNA moietythrough a coupled redox conducting polymer to an electrode.

Suitable mediators include water soluble ferrocene/ferriciniumhydroquinones/quinones, reducible and oxidizable components of organicsalts, cobaltocenes, the hexa- and octacyanides of molybdenum, tungstenand iron. In addition, macrocycles and chelating ligands of transitionmetals such as cobalt, ruthenium and nickel are used, includingCo(ethylenediamine)₃ and Ru(ethylenediamine)₃ and the trisbypyridyl andhexamine complexes of transition metals such as Co, Ru, Fe, and Os (seeAlyanasundaram, supra).

In a preferred embodiment, electron transport between the electrode andthe nucleic acid can be direct via a covalent bond. One advantage ofthese systems is that the orientation of the DNA probe can be influencedto reduce any bending back of the probe onto the electrode. Also, moreprecise control of applied potential and measured current is associatedwith short covalent linkages versus gels and polymers.

In a preferred embodiment, the covalent bonds must be highly conductingsuch as in a redox polymer (Hellar, A. Acc. Chem. Res. Vol. 23, p. 128,1990). Alternatively, if they are poorly conducting, the length of thelinkage must be kept short. Accordingly, a preferred embodiment has anelectron traversing no more than about five σ bonds, with no more thanthree being especially preferred. Carbon paste and glassy carbon rodshave proven reliable and effective as electrodes in a variety ofchemical sensors, including sensitive glucose oxidase enzyme-basedbiosensors, and may be used in the present invention. In addition,flexible siloxane and ethylene oxide polymers covalently attached toferrocene or Os(bpy)₂ molecules have been shown to be highly effectiveredox polymers for mediating electron transfer from several enzymes toan electrode. Amino-ribose modified nucleic acids are attached to carbonelectrodes by variations of these literature techniques. Finally,nucleic acids are more directly attached to oxidized carbon electrodesvia guanosine residues, using known carbodiimide andN-hydroxysuccinimide chemistry.

In a preferred embodiment, glassy carbon electrodes (GCEs) are used. Inthis embodiment, amine groups such as outlined above on the 2′ or 3′carton of the ribose ring are used for attachment. The reaction proceedsvia the oxidation of an amine group to a cation radical which forms achemically stable and covalent bond between the amine and the edge planeof the GCE surface (see Deinhammer, R, et al. Langmuir 10: 1306 (1994))This synthetic approach has been well characterized using X-rayphoto-electron spectroscopy and cyclic voltammetry. The yield using thischemistry can be quite high, approximately 1×10¹⁰ molecules/cm². Theamine compound forms a stable bond to the carbon surface, and stericeffects influence binding efficiency. The reactivity of primary aminesis substantially higher than secondary amines; the binding of tertiaryamines is not observed at all.

Employing the amino-modified (primary amine group) oligonucleotidesdescribed earlier, the procedure developed by Deinhammer, R, et al. toprepare the GCEs for electrochemical treatment in amine containingsolution is depicted in FIG 8A.

In addition, DNA has been immobilized onto GCEs using a water solublecarodimide (Mikkelsen et al., Electroanalysis 4:929 (1992)).

In a preferred embodiment, the nucleic acids of the invention areattached to gold electrodes. Several methods are available for thecovalent attachment of redox active species to gold surfaces andelectron transfer reactions with these materials have been observed.Hydroxy thiols (OH(CH₂)_(x)SH) of varying lengths are prepared byvariation of literature procedures (see Miller, C. et al. J. Phys. Chem.95: 877 (1991) and Chidsey, C. E. D., Science, V. 251, p. 919, (1991)).Example 8 outlines the preparation of hydroxyl thiols which are attachedto gold electrodes.

Alternative procedures for the preparation of hydroxythiols are known inthe art. Au electrodes or surfaces are prepared by literature proceduresand the modified hydroxythiols adsorbed onto the Au.

In an additional embodiment, the modified nucleic acids of the inventionare covalently attached to thin film oxidized surfaces. It has beenreported that a variety of compounds can be covalently bonded (in theform of monolayers) to thin-film SnO₂, TiO₂, and RuO₂ and Pt electrodes(see Lenhard, J. and Murray, R. J. Electroanal. Chem. 78:195 (1977)).Reversible electrochemistry of surface bound complexes such as3,5-dinitrobenzamide to electrodes has been observed. The reportedcomplexes are attached to the electrode via an amide bond linkage.Employing these literature procedures, analogous derivatives usingamino-modified oligonucleotides described in this work can be preparedand are schematically represented in FIG. 8B.

Accordingly, using the above methods, oligonucleotides may be attachedto a solid support such that the electrode serves as either the electrondonor moiety or the electron acceptor moiety.

Thus, all combinations of electron donors and acceptors may be made: twotransition metal complexes; two organic electron transfer species; onetransition metal, one organic moiety; one transition metal and anelectrode; and one organic moiety and an electrode. The choice of theelectron transfer species will depend in part on the method ofinitiation and detection required, as is more fully described below.

The term “target sequence” or grammatical equivalents herein means anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,cDNA, mRNA, or others. It may be any length, with the understanding thatlonger sequences are more specific. As is outlined more fully below,probes are made to hybridize to target sequences to determine thepresence or absence of the target sequence in a sample. Generallyspeaking, this term will be understood by those skilled in the art.

The probes of the present invention are designed to be complementary tothe target sequence, such that hybridization of the target sequence andthe probes of the present invention occurs. As outlined below, thiscomplementarity need not be perfect; there may be any number of basepair mismatches which will interfere with hybridization between thetarget sequence and the single stranded nucleic acids of the presentinvention. However, if the number of mutations is so great that nohybridization can occur under even the least stringent of hybridizationconditions, the sequence is not a complementary target sequence.

A variety of hybridization conditions may be used in the presentinvention. As is known in the art, “high” stringency usually refers toconditions such as 0.1×SSC at 65° C., reduced stringency conditionsinclude 2-5×SSC at 25-50° C. The hybridization conditions may also varywhen a non-ionic backbone such as PNA is used, as is known in the art.

The terms “first target domain” and “second target domain” orgrammatical equivalents herein means two portions of a target sequencewithin a nucleic acid which is under examination. The first targetdomain may be directly adjacent to the second target domain, or thefirst and second target domains may be separated by an interveningtarget domain. The terms “first” and “second” are not meant to confer anorientation of the sequences with respect to the 5′-3′ orientation ofthe target sequence. For example, assuming a 5′-3′ orientation of thecomplementary target sequence, the first target domain may be locatedeither 5′ to the second domain, or 3′ to the second domain.

The present invention is directed, in part, to the site-selectivemodification of nucleic acids with redox active moieties such astransition metal complexes for the preparation of a new series ofbiomaterials capable of long distance electron transfer through anucleic acid matrix. The present invention provides for the preciseplacement of electron transfer donor and acceptor moieties atpredetermined sites on a single stranded or double stranded nucleicacid. In general, electron transfer between electron donor and acceptormoieties in a double helical nucleic acid does not occur at anappreciable rate unless nucleotide base pairing exists in the sequencebetween the electron donor and acceptor in the double helical structure.

This differential in the rate of electron transfer forms the basis of autility of the present invention for use as probes. In the system of thepresent invention, where electron transfer moieties are covalently boundto the backbone of a nucleic acid, the electrons putatively travel viathe π-orbitals of the stacked base pairs of the double stranded nucleicacid. The electron transfer rate is dependent on several factors,including the distance between the electron donor-acceptor pair, thefree energy (ΔG) of the reaction, the reorganization energy (λ), thecontribution of the intervening medium, the orientation and electroniccoupling of the donor and acceptor pair, and the hydrogen bondingbetween the bases.

The contribution of the intervening medium depends, in part, on thenumber of sigma (σ) bonds the electron must traverse from the electrondonor to reach the bases stack, or to exit the stack to reach theelectron acceptor. As is shown in FIG. 5, when the metal is bound to theribose-phosphate backbone via an amine moiety at the 2′ carbon of theribose, an electron must travel through four σ bonds to reach the stack:the metal to nitrogen bond, the nitrogen to 2′ carbon bond, and from the2′ carbon to the base, or vice versa depending on the direction of theelectron flow. Since the base of the nucleotide is conjugated in somedegree, the base can be considered to be the edge of the “π-way”; thatis, the conjugated π orbitals of the stacked base pairs. When the metalis bound to the ribose-phosphate backbone via the 3′ carbon of theribose, an electron must traverse through 5 σ bonds. When the metal isbound via phosphoramide-type linkages, an electron must traverse through7 σ bonds. In the preferred embodiments, the compositions of theinvention are designed such that the electron transfer moieties are asclose to the “pi-way” as possible without significantly disturbing thesecondary and tertiary structure of the double helical nucleic acid,particularly the Watson-Crick basepairing.

The effect on the electron transfer rate by the hydrogen bonding betweenthe bases is a dependence on the actual nucleic acid sequence, since A-Tpairs contain one less hydrogen bond than C-G pairs. However, thissequence dependence is overshadowed by the determination that there is ameasurable difference between the rate of electron transfer within a DNAbase-pair matrix, and the rate through the ribose-phosphate backbone,the solvent or other electron tunnels. This rate differential is thoughtto be at least several orders of magnitude, and may be as high as fourorders of magnitude greater through the stacked nucleotide bases ascompared to other electron transfer pathways. Thus the presence ofdouble stranded nucleic acids, for example in gene probe assays, can bedetermined by comparing the rate of electron transfer for theunhybridized probe with the rate for hybridized probes.

In one embodiment, the present invention provides for novel gene probes,which are useful in molecular biology and diagnostic medicine. In thisembodiment, single stranded nucleic acids having a predeterminedsequence and covalently attached electron donor and electron acceptormoieties are synthesized. The sequence is selected based upon a knowntarget sequence, such that if hybridization to a complementary targetsequence occurs in the region between the electron donor and theelectron acceptor, electron transfer proceeds at an appreciable anddetectable rate. Thus, the present invention has broad general use, as anew form of labeled gene probe. In addition, since detectable electrontransfer in unhybridized probes is not appreciable, the probes of thepresent invention allow detection of target sequences without theremoval of unhybridized probe. Thus, the present invention is uniquelysuited to automated gene probe assays or field testing.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis may also bedetected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, enterotoxic strains of E. coli, andLegionnaire's disease bacteria. Similarly, bioremediation strategies maybe evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

The present invention also finds use as a unique methodology for thedetection of mutations in target nucleic acid sequences. As a result, ifa single stranded nucleic acid containing electron transfer moieties ishybridized to a target sequence with a mutation, the resultingperturbation of the base pairing of the nucleosides will measurablyaffect the electron transfer rate. This is the case if the mutation is asubstitution, insertion or deletion. Alternatively, two single strandednucleic acids each with a covalently attached electron transfer speciesthat hybridize adjacently to a target sequence may be used. Accordingly,the present invention provides for the detection of mutations in targetsequences.

Thus, the present invention provides for extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

In an alternate embodiment double stranded nucleic acids have covalentlyattached electron donor and electron acceptor moieties on oppositestrands. Such nucleic acids are useful to detect successful geneamplification in polymerase chain reactions (PCR), thus allowingsuccessful PCR reactions to be an indication of the presence or absenceof a target sequence. PCR may be used in this manner in several ways.For example, if one of the two PCR primers contains a 5′ terminallyattached electron donor, and the other contains a 5′ terminally attachedelectron acceptor, several rounds of PCR will generate doubly labeleddouble stranded fragments (occasionally referred to as “amplicons”).After appropriate photoinduction, the detection of electron transferprovides an indication of the successful amplification of the targetsequence as compared to when no amplification occurs. A particularadvantage of the present invention is that the separation of the singlestranded primers from the amplified double stranded DNA is notnecessary, as outlined above for probe sequences which contain electrontransfer moieties. Alternatively, the detection of a target sequence viaPCR is done by attaching one electron transfer moiety species to one orboth of the primers. The other electron transfer moiety species isattached to individual nucleosides of the PCR reaction pool, as isdescribed herein. Incorporation of the nucleosides containing theelectron transfer moiety into the nucleic acid during the PCR reactionresults in both electron transfer species being attached either to thesame single strand or to opposite strands, or both. Allowing the newlysynthesized nucleic acid to remain in a hybridized form allows thedetection of successful elongation via electron transfer, and thus thedetection of a target sequence. In this way, the present invention isused for PCR detection of target sequences

In another embodiment the present invention provides for double strandednucleic acids with covalently attached electron donor and electronacceptor moieties to serve as bioconductors or “molecular wire”. Theelectron transport may occur over distances up to and in excess of 28 Åper electron donor and acceptor pair. In addition, the rate of electrontransfer is very fast, even though dependent on the distance between theelectron donor and acceptor moieties. By modifying the nucleic acid inregular intervals with electron donor and/or electron acceptor moieties,it may be possible to transport electrons over long distances, thuscreating bioconductors. These bioconductors are useful in a large numberof applications, including traditional applications for conductors suchas mediators for electrochemical reactions and processes.

In addition, these bioconductors may be useful as probes forphotosynthesis reactions as well as in the construction of syntheticlight harvesting systems. The current models for the electron transfercomponent of an artificial light harvesting system have severalproblems, as outlined above, including a dependence on solvent polarityand composition, and a lack of sufficient rigidity without arduoussynthesis. Thus the present invention is useful as both a novel form ofbioconductor as well as a novel gene probe.

The present invention provides nucleic acids with covalently attachedelectron transfer moieties. The electron transfer moieties may beattached to the nucleic acid at a variety of positions.

In one embodiment, the electron donor and acceptor moieties are added tothe 3′ and/or 5′ termini of the nucleic acid on either thesugar-phosphate backbone or a terminal base. In alternative embodiments,the electron donor and acceptor moieties are added to the backbone ofone or more internal nucleosides, that is, any nucleoside which is notthe 3′ or 5′ terminal nucleoside. In a further embodiment, the electrondonor and acceptor moieties are added to the backbone of both internaland terminal nucleosides.

In a preferred embodiment, the electron transfer moieties are attachedto the ribose-phosphate backbone in a number of positions. As shown inFIG. 5, several positions are possible, with attachment to a ribose ofthe ribose-phosphate backbone being particularly preferred. Accordingly,in FIG. 5, the most preferred site of attachment of a electron transfermoiety is M₁, followed by M₄, M₂ and M₃, in that order. In a preferredembodiment, the electron transfer moieties are attached at the 2′ or 3′position on the ribose, with 2′ being particularly preferred.

In a preferred embodiment, the electron transfer moieties do notintercalate, and are attached such that do not intercalate. Thus, whileit is possible to utilize a “linker”, such as alternating double bondsto attach the electron transfer moiety to the nucleic acid, the linkeris either preferably not longer than the equivalent of one or twonucleosides in length, or is not significantly flexible to allowintercalation. Preferably, if linkers are used, they are attached viathe ribose of the nucleic acid backbone.

In one embodiment, the electron transfer moieties are added to the basesof the terminal nucleosides. Thus, when the target sequence to bedetected is n nucleosides long, a probe can be made which has an extraterminal nucleoside at one or both of the ends of the nucleic acid (n+1or n+2), which are used to covalently attach the electron transfermoieties but which do not participate in basepair hybridization. Thisextra terminal nucleoside is important since attachment of electrontransfer moieties to an internal nucleoside base is expected to perturbWatson-Crick basepairing. That is, the base used for covalent attachmentshould be outside of the region used to identify the target sequence.Additionally, it is preferred that upon probe hybridization, theterminal nucleoside containing the electron transfer moiety covalentlyattached at the base be directly adjacent to Watson-Crick basepairednucledsides; that is, the electron transfer moiety should be as close aspossible to the stacked π-orbitals of the bases such that an electrontravels through a minimum of σ bonds to reach the “π-way”, oralternatively can otherwise electronically contact the π-way.

In one embodiment, a single stranded nucleic acid is labeled with anelectron transfer moiety via the terminal bases at both ends. Alternateembodiments utilize a terminal base and a 5′ or a 3′ terminalribose-phosphate attachment as described above. In further embodiments,compositions are provided comprising a first single stranded nucleicacid containing an electron donor covalently attached at a terminal baseand a second single stranded nucleic acid containing an electronacceptor covalently attached at a position as described above, that is,at a 5′, 3′ or internal position; alternatively, the electron donor andacceptor may be switched. A particularly preferred embodiment utilizesan electrode as one of the electron transfer moieties with the otherelectron transfer moiety being attached to a terminal base, preferablyon the same single strand.

The present invention further provides methods For the site-specificaddition of electron transfer moieties to nucleic acids. As outlinedabove, the electron transfer moieties may be added at the 2′ or 3′position of a ribose of the ribose-phosphate backbone, to a 3′ or 5′terminal base, or to an internal nucleoside using peptide nucleic acidlinkages, phosphoramidate bonds, phosphorothioate bonds,phosphorodithioate bonds, or O-methyl phosphoramidate bonds. Molecularmechanics calculations indicate that perturbations due to themodification of at the ribose of the terminal nucleosides of nucleicacids are minimal, and Watson-Crick base pairing is not disrupted(unpublished data using Biograf from Molecular Simulations Inc., SanDiego, Calif.).

For attachment to a ribose, a preferred embodiment utilizes modifiednucleosides to attach the electron transfer moieties. Preferablyamino-modified nucleosides and nucleosides are used. In an alternateembodiment, thio-modified nucleosides are used to attach the electrontransfer moieties of the invention.

The modified nucleosides are then used to site-specifically add atransition metal electron transfer moiety, either to the 3′ or 5′termini of the nucleic acid, or to any internal nucleoside. Either the2′ or 3′ position of the ribose may be altered for attachment at the 3′terminus; for attachment to an internal ribose or the 5′ terminus, the2′ position is preferred. Thus, for example, the 2′ position of theribose of the deoxyribo- or ribonucleoside is modified prior to theaddition of the electron transfer species, leaving the 3′ position ofthe ribose unmodified for subsequent chain attachment if necessary. In apreferred embodiment, an amino group is added to the 2′or 3′ carbon ofthe sugar using established chemical techniques. (Imazawa et al., J.Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem. 42(4):714(1977); Verheyden et al. J. Org. Chem. 36(2):250 (1971)).

The amino-modified nucleosides made as described above are converted tothe 2′ or 3′ modified nucleotide triphosphate form using standardbiochemical methods (Fraser et al., Proc. Natl. Acad. Sci. USA, 4:2671(1973)).

Modified nucleosides for the attachment of the electron transfermoieties to the bases, is done as outlined in Telser, supra, both ofwhich are expressly incorporated by reference. These modifiednucleosides are then incorporated at either the 3′ or 5′ terminus asoutlined below.

Once the modified nucleosides are prepared, protected and activated,they may be incorporated into a growing oligonucleotide by standardsynthetic techniques (Gait, Oligonucleotide Synthesis: A PracticalApproach, IRL Press, Oxford, UK 1984; Eckstein) in several ways. In oneembodiment, one or more modified nucleosides are incorporated into agrowing oligonucleotide chain by using standard molecular biologytechniques such as with the use of the enzyme DNA polymerase I, T4 DNApolymerase, T7 DNA polymerase, Taq DNA polymerase, reversetranscriptase, and RNA polymerases. For the incorporation of a 3′modified nucleoside to a nucleic acid, terminaldeoxynucleotidyltransferase may be used. (Ratliff, Terminaldeoxynucleotidyltransferase. In The Enzymes, Vol 14A. P. D. Boyer ed. pp105-118. Academic Press, San Diego, Calif. 1981). Alternatively, andpreferably, the amino nucleoside is converted to the phosphoramidite orH-phosphonate form, which are then used in solid-phase or solutionsyntheses of oligonucleotides. In this way the modified nucleoside,either for attachment at the ribose (i.e. amino- or thiol-modifiednucleosides) or the base, is incorporated into the oligonucleotide ateither an internal position or the 5′ terminus. This is generally doneby protecting the 5′ position of the ribose with 4′,4-dimethoxytrityl(DMT) followed by reaction with2-cyanoethoxy-bis-diisopropylaminophosphine in the presence ofdiisopropylammonium tetrazolide to give the phosphoramidite as is knownin the art; although other techniques may be used as will be appreciatedby those in the art. See Gait, supra; Caruthers, Science 230:281 (1985),both of which are expressly incorporated herein by reference.

For attachment of an electron transfer moiety to the 3′ terminus, apreferred method utilizes the attachment of the modified nucleoside tocontrolled pore glass (CPG) or other polymeric supports. In thisembodiment, the modified nucleoside is protected at the 5′ end with DMT,and then reacted with succinic anhydride with activation. The resultingsuccinyl compound is attached to CPG or other polymeric supports as isknown in the art. Further phosphoramidite nucleosides are added, eithermodified or not, to the 5′ end after deprotection.

In other embodiments, the electron transfer moiety or moieties are addedto the middle of the nucleic acid, i.e. to an internal nucleoside. Thismay be accomplished in three ways.

In a preferred embodiment, a modified nucleoside is incorporated at the5′ terminus as described above. In this embodiment, oligonucleotidesynthesis simply extends the 5′ end from the modified nucleoside usingstandard techniques. This results in an internally amino modifiedoligonucleotide.

In one embodiment, the nucleosides are modified to contain an aromaticamine capable of binding an electron transfer moiety at either the 2′ or3′ position of the ribose. For example, one of the nitrogens ofimidazole can be attached at the 2′ or 3′ position of the ribose andthus used to attach the electron transfer moiety such as a transitionmetal complex. This may effectively reduce the number of σ bonds anelectron must travel through to reach the “pi-way” since the imidazoleoffers substantially less resistance to electron transfer as compared toa σ bond. In a preferred embodiment, the imidazole is attached at the 2′position of the ribose. In an alternate embodiment, the imidazole isattached at the 3′ position. The imidazole-modified nucleoside may beincorporated into an oligonucleotide as outlined herein foramino-modified nucleosides.

In an alternate embodiment, electron transfer moieties are added to thebackbone at a site other than ribose, resulting in an internalattachment. For example, phosphoramide rather than phosphodiesterlinkages can be used as the site for transition metal modification.These transition metals serve as the donors and acceptors for electrontransfer reactions. While structural deviations from nativephosphodiester linkages do occur and have been studied using CD and NMR(Heller, Acc. Chem. Res. 23:128 (1990); Schuhmann et al. J. Am. Chem.Soc. 113:1394 (1991)), the phosphoramidite internucleotide link has beenreported to bind to complementary polynucleotides and is stable(Beaucage et al., supra, and references therein; Letsinger, supra;Sawai, supra; Jager, Biochemistry 27:7237 (1988)). In this embodiment,dimers of nucleotides are created with phosphoramide linkages at eitherthe 2′-5′ or 3′-5′ positions. A preferred embodiment utilizes the 3′-5′position for the phosphoramide linkage, such that structural disruptionof the subsequent Watson-Crick basepairing is minimized. These dimerunits are incorporated into a growing oligonucleotide chain, as above,at defined intervals, as outlined below.

Thus, the present invention provides methods for making a nucleic acidwith covalently attached electron transfer moieties. In a preferredembodiment, the method is for making a nucleic acid with an electrontransfer moiety attached at the 3′ terminus of said nucleic acid. Themethod comprises attaching a 2′-amino modified nucleoside to controlpore glass, and adding phosphoramidite nucleosides to the 5′ terminus ofthe modified nucleoside to form a nucleic acid. The nucleic acid is thenoptionally cleaved from the CPG using known methods. The nucleic acidmay be hybridized to its complement, to protect the bases frommodification, if required, and the electron transfer moiety is added tothe 2′-amino modified nucleoside.

In a preferred embodiment, methods for making a nucleic acid with anelectron transfer moiety attached at the 5′ terminus are provided. Themethod comprises attaching a nucleoside to control pore glass, andadding phosphoramidite nucleosides to the 5′ terminus of the nucleosideto form a nucleic acid. A 2′ or 3′ amino modified nucleoside is added tothe 5′ terminus, and the nucleic acid is optionally cleaved from theCPG. The nucleic acid may be hybridized to its complement if required,and the electron transfer moiety is added to the 2′ or 3′-amino modifiednucleoside.

In a preferred embodiment, a method for making a single stranded nucleicacid with electron transfer moieties attached at both the 3′ and 5′terminus. The method comprises attaching a modified nucleoside tocontrol pore glass. The modified nucleoside may be eitheramino-modified, for attachment via the ribose as described herein, ormodified at the base. Additional phosphoramidite nucleosides are addedto the 5′ terminus of the modified nucleoside to form a nucleic acid. Amodified phosphoramidite nucleoside is further added to the 5′ terminusof the nucleic acid, which is then optionally cleaved off the controlpore glass and may be hybridized to its complement. An electron donormoiety is added to one modified nucleoside and an electron acceptormoiety is added to the other modified nucleoside.

The cleavage from the CPG may occur either prior to transition metalmodification or afterwards.

It should be understood that it is important that the basepairing of thenucleoside bases is not significantly perturbed in order to allowhybridization, good electron transfer rates, and the detection ofmismatches. Thus, for example, the transition metal moieties, whenattached to the nucleic acids of the invention, do not intercalate, i.e.insert and stack between the basepairs of the double stranded nucleicacid. Intercalation of the transition metals with the accompanyingligands disturbs the basepairing, and thus hinders the transfer ofelectrons and the identification of mismatches. Similarly, with theexception of terminal bases, as is outlined below, attaching thetransition metal complexes at the nucleoside bases (Telser et al.,supra) also disturbs the basepairing and impedes the identification ofmismatches.

It should be noted that when using the above techniques for themodification of internal residues it is possible to create a nucleicacid that has an electron transfer species on the next-to-last 3′terminal nucleoside, thus eliminating the need for the extra stepsrequired to produce the 3′ terminally labeled nucleoside.

In a further embodiment for the modification of internal residues, 2′ or3′ modified nucleoside triphosphates are generated using the techniquesdescribed above for the 3′ nucleoside modification. The modifiednucleosides are inserted internally into nucleic acid using standardmolecular biological techniques for labeling DNA and RNA. Enzymes usedfor said labelling include DNA polymerases such as polymerase I, T4 DNApolymerase, T7 DNA polymerase, Taq DNA polymerase, reverse transcriptaseand RNA polymerases such as E. coli RNA polymerase or the RNApolymerases from phages SP6, T7 or T3 (Short Protocols in MolecularBiology, 1992. Ausubel et al. Ed. pp 3.11-3.30).

As described above, the electron transfer moiety, preferably atransition metal complex, may be attached to any of the five bases(adenine, thymine, uracil, cytosine, guanine and other non-naturallyoccurring bases such as inosine, xanthine, and hypoxanthine, amongothers). This is done using well known techniques; see Telser et al., J.Am. Chem. Soc. 111:7226-7232 (1989); Telser et al., J. Am. Chem. Soc.111:7221-7226 (1989). As outlined herein, these terminally modifiednucleosides may be attached to the nucleic acid enzymatically as isknown in the art, using DNA polymerases; alternatively, the modifiednucleosides may be incorporated into a growing oligonucleotide chainusing traditional phosphoramidite chemistry during oligonucleotidesynthesis as is outlined herein.

The exposed amine or other ligand at the 2′ or 3′ position of theribose, the phosphoramide linkages, or the other linkages useful in thepresent invention, are readily modified with a variety of electrontransfer moieties, and particularly transition metal complexes withtechniques readily known in the art (see for example Millet et al, inMetals in Biological Systems, Sigel et al. Ed. Vol. 27, pp 223-264,Marcell Dekker Inc. New York, 1991 and Durham, et al. in ACS Advances inChemistry Series, Johnson et al. Eds., Vol. 226, pp 180-193, AmericanChemical Society, Washington D.C.; and Meade et al., J. Am. Chem. Soc.111:4353 (1989)). Generally, these techniques involve contacting apartially chelated transition metal complex with the amine group of themodified nucleoside.

The organic electron transfer species are also added to the functionalgroup of the modified nucleoside such as an amine group, usingtechniques known in the art.

When peptide nucleic acids (PNA) are used, attachment of the electrontransfer moieties proceeds as follows. The amino group at the N-terminusof the PNA will bind a partially chelated transition metal or organicelectron transfer moiety similar to the amino-modified ribose. Additionto the carboxy terminus can proceed in a variety of ways, one of whichis depicted in FIG. 7. Additionally, for single stranded PNAs, oneelectron transfer moiety may be attached to the N-terminus, and theother electron transfer moiety is attached to the terminal base at thecarboxy terminus. Alternatively, both transfer moieties are attached toterminal bases. Similar combinations may be made for two single strandednucleic acids, each containing an electron transfer moiety.

In addition, the present invention provides a novel method for the sitespecific addition to the ribose-phosphate backbone of a nucleic acid ofelectron donor and electron acceptor moieties to a previously modifiednucleoside.

In one embodiment, the electron donor and acceptor moieties are attachedto the modified nucleoside by methods which utilize a unique protectivehybridization step. In this embodiment, the modified single strandnucleic acid is hybridized to an unmodified complementary sequence. Thisblocks the sites on the heterocyclic bases that are susceptible toattack by the transition metal electron transfer species.

When the terminal bases are to be labeled with electron transferspecies, the complementary sequence does not extend to the base to belabeled. That is, a complementary sequence of n nucleosides in length ischosen for hybridization to a probe sequence of n+1 or n+2, such thatthe terminal base is not protected. Thus the unprotected base is exposedto the electron transfer moiety such that the moiety is attached to thebase.

After successful addition of the desired metal complex, the modifiedduplex nucleic acid is separated into single strands using techniqueswell known in the art.

In a preferred embodiment, single stranded nucleic acids are made whichcontain one electron donor moiety and one electron acceptor moiety. Theelectron donor and electron acceptor moieties may be attached at eitherthe 5′ or 3′ end of the single stranded nucleic acid. Alternatively, theelectron transfer moieties may be attached to internal nucleosides, orone to an internal nucleoside and one to a terminal nucleoside. Itshould be understood that the orientation of the electron transferspecies with respect to the 5′-3′ orientation of the nucleic acid is notdeterminative. Thus, as outlined in FIG. 1, any combination of internaland terminal nucleosides may be utilized in this embodiment.

In an alternate preferred embodiment, single stranded nucleic acids withat least one electron donor moiety and at least one electron acceptormoiety are used to detect mutations in a complementary target sequence.A mutation, whether it be a substitution, insertion or deletion of anucleoside or nucleosides, results in incorrect base pairing in ahybridized double helix of nucleic acid. Accordingly, if the path of anelectron from an electron donor moiety to an electron acceptor moietyspans the region where the mismatch lies, the electron transfer will beeliminated or reduced such that a change in the relative rate will beseen. Therefore, in this embodiment, the electron donor moiety isattached to the nucleic acid at a 5′ position from the mutation, and theelectron acceptor moiety is attached at a 3′ position, or vice versa.

In this embodiment it is also possible to use an additional label on themodified single stranded nucleic acid to detect hybridization wherethere is one or more mismatches. If the complementary target nucleicacid contains a mutation, electron transfer is reduced or eliminated. Toact as a control, the modified single stranded nucleic acid may beradio- or fluorescently labeled, such that hybridization to the targetsequence may be detected, according to traditional molecular biologytechniques. This allows for the determination that the target sequenceexists but contains a substitution, insertion or deletion of one or morenucleosides. Alternatively, single stranded nucleic acids with at leastone electron donor moiety and one electron acceptor moiety whichhybridize to regions with exact matches can be used as a controls forthe presence of the target sequence.

It is to be understood that the rate of electron transfer through adouble stranded nucleic acid helix depends on the nucleoside distancebetween the electron donor and acceptor moieties. Longer distances willhave slower rates, and consideration of the rates will be a parameter inthe design of probes and bioconductors. Thus, while it is possible tomeasure rates for distances in excess of 100 nucleosides, a preferredembodiment has the electron donor moiety and the electron acceptormoiety separated by at least 3 and no more than 100 nucleosides. Morepreferably the moieties are separated by 8 to 64 nucleosides, with 15being the most preferred distance.

In addition, it should be noted that certain distances may allow theutilization of different detection systems. For example, the sensitivityof some detection systems may allow the detection of extremely fastrates; i.e. the electron transfer moieties may be very close together.Other detection systems may require slightly slower rates, and thisallow the electron transfer moieties to be farther apart.

In an alternate embodiment, a single stranded nucleic acid is modifiedwith more than one electron donor or acceptor moiety. For example, toincrease the signal obtained from these probes, or decrease the requireddetector sensitivity, multiple sets of electron donor-acceptor pairs maybe used.

As outlined above, in some embodiments different electron transfermoieties are added to a single stranded nucleic acid. For example, whenan electron donor moiety and an electron acceptor moiety are to beadded, or several different electron donors and electron acceptors, thesynthesis of the single stranded nucleic acid proceeds in several steps.First partial nucleic acid sequences are made, each containing a singleelectron transfer species, i.e. either a single transfer moiety orseveral of the same transfer moieties, using the techniques outlinedabove. Then these partial nucleic acid sequences are ligated togetherusing techniques common in the art, such as hybridization of theindividual modified partial nucleic acids to a complementary singlestrand, followed by ligation with a commercially available ligase.

Alternatively, single stranded nucleic acid may be made by incorporatingan amino modified nucleoside at two positions using the abovetechniques. As a result of the synthesis, one of the amino modifiednucleosides has a temporary protecting group on the amine such as DMT.Upon hybridization to the complementary unmodified strand, theunprotected amine is exposed to the first electron transfer moiety, i.e.either a donor or an acceptor, resulting in covalent attachment. Theprotecting group of the protected amino-modified nucleoside is thenremoved, and the hybrid is contacted with the second electron transferspecies, and the strands separated, resulting in a single strand beinglabeled with both a donor and acceptor. The single strand containing theproper electron transfer moieties is then purified using traditionaltechniques.

In a preferred embodiment, single stranded nucleic acids are made whichcontain one electron donor moiety or one electron acceptor moiety. Theelectron donor and electron acceptor moieties are attached at either the5′ or 3′ end of the single stranded nucleic acid. Alternatively, theelectron transfer moiety is attached to an internal nucleoside.

It is to be understood that different species of electron donor andacceptor moieties may be attached to a single stranded nucleic acid.Thus, more than one type of electron donor moiety or electron acceptormoiety may be added to any single stranded nucleic acid.

In a preferred embodiment, a first single stranded nucleic acid is madewith on or more electron donor moieties attached. A second singlestranded nucleic acid has one or more electron acceptor moietiesattached. In this embodiment, the single stranded nucleic acids are madefor use as probes for a complementary target sequence. In oneembodiment, the complementary target sequence is made up of a firsttarget domain and a second target domain, where the first and secondsequences are directly adjacent to one another. In this embodiment, thefirst modified single stranded nucleic acid, which contains onlyelectron donor moieties or electron acceptor moieties but not both,hybridizes to the first target domain, and the second modified singlestranded nucleic acid, which contains only the corresponding electrontransfer species, binds to the second target domain. The relativeorientation of the electron transfer species is not important, asoutlined in FIG. 2, and the present invention is intended to include allpossible orientations.

In the design of probes comprised of two single stranded nucleic acidswhich hybridize to adjacent first and second target sequences, severalfactors should be considered. These factors include the distance betweenthe electron donor moiety and the electron acceptor moiety in thehybridized form, and the length of the individual single strandedprobes. For example, it may be desirable to synthesize only 5′terminally labeled probes. In this case, the single stranded nucleicacid which hybridizes to the first sequence may be relatively short,such that the desirable distance between the probes may be accomplished.For example, if the optimal distance between the electron transfermoieties is 15 nucleosides, then the first probe may be 15 nucleosideslong.

In one aspect of this embodiment, the two single stranded nucleic acidswhich have hybridized to the adjacent first and second target domainsare ligated together prior to the electron transfer reaction. This maybe done using standard molecular biology techniques utilizing a DNAligase, such as T4 DNA ligase.

In an alternative embodiment, the complementary target sequence willhave a first target domain, an intervening target domain, and a secondtarget domain. In this embodiment, the first modified single strandednucleic acid, which contains only electron donor moieties or electronacceptor moieties but not both, hybridizes to the first target domain,and the second modified single stranded nucleic acid, which containsonly the corresponding electron transfer species, binds to the secondtarget domain. When an intervening single stranded nucleic acidhybridizes to the intervening target sequence, electron transfer betweenthe donor and acceptor is possible. The intervening sequence may be anylength, and may comprise a single nucleoside. Its length, however,should take into consideration the desirable distances between theelectron donor and acceptor moieties on the first and second modifiednucleic acids. Intervening sequences of lengths greater than 14 aredesirable, since the intervening sequence is more likely to remainhybridized to form a double stranded nucleic acid if longer interveningsequences are used. The presence or absence of an intervening sequencecan be used to detect insertions and deletions.

In one aspect of this embodiment, the first single stranded nucleic acidhybridized to the first target domain, the intervening nucleic acidhybridized to the intervening domain, and the second single strandednucleic acid hybridized to the second target domain, may be ligatedtogether prior to the electron transfer reaction. This may be done usingstandard molecular biology techniques. For example, when the nucleicacids are DNA, a DNA ligase, such as T4 DNA ligase can be used.

The complementary target single stranded nucleic acid of the presentinvention may take many forms. For example, the complementary targetsingle stranded nucleic acid sequence may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. One skilled in theart of molecular biology would understand how to construct useful probesfor a variety of target sequences using the present invention.

In one embodiment, two single stranded nucleic acids with covalentlyattached electron transfer moieties have complementary sequences, suchthat they can hybridize together to form a bioconductor. In thisembodiment, the hybridized duplex is capable of transferring at leastone electron from the electron donor moiety to the electron acceptormoiety. In a preferred embodiment, the individual single strandednucleic acids are aligned such that they have blunt ends; in alternativeembodiments, the nucleic acids are aligned such that the double helixhas cohesive ends. In either embodiment, it is preferred that there beuninterrupted double helix base-pairing between the electron donormoiety and the electron acceptor moiety, such that electrons may travelthrough the stacked base pairs.

In one bioconductor embodiment, the double stranded nucleic acid has onesingle strand nucleic acid which carries all of the electron transfermoieties.

In another embodiment, the electron transfer moieties may be carried oneither strand, and in any orientation. For example, one strand may carryonly electron donors, and the other only electron acceptors or bothstrands may carry both.

In one embodiment, the double stranded nucleic acid may have differentelectron transfer moieties covalently attached in a fixed orientation,to facilitate the long range transfer of electrons. This type of systemtakes advantage of the fact that electron transfer species may act asboth electron donors and acceptors depending on their oxidative state.Thus, an electron donor moiety, after the loss of an electron, may actas an electron acceptor, and vice versa. Thus, electron transfermoieties may be sequentially oriented on either strand of the doublestranded nucleic acid such that directional transfer of an electron oververy long distances may be accomplished. For example, a double strandednucleic acid could contain a single electron donor moiety at one end andelectron acceptor moieties, of the same or different composition,throughout the molecule. A cascade effect of electron transfer could beaccomplished in this manner, which may result in extremely long rangetransfer of electrons. This may be accomplished, for example, byincorporating transition metal complexes that possess a range inoxidation potentials due to ligand substitutions made at the metalcenter.

The choice of the specific electron donor and acceptor pairs will beinfluenced by the type of electron transfer measurement used; for areview, see Winkler et al., Chem. Rev. 92:369-379 (1992). When along-lived excited state can be prepared on one of the redox sites,direct measurement of the electron transfer rate after photoinductioncan be measured, using for example the flash-quench method of Chang etal., J. Amer. Chem. Soc. 113:7057 (1991). In this preferred embodiment,the excited redox site, being both a better acceptor and donor than theground-state species, can transfer electrons to or from the redoxpartner. An advantage of this method is that two electron transfer ratesmay be measured: the photoinduced electron transfer rates and thermalelectron-hole recombination reactions. Thus differential rates may bemeasured for hybridized nucleic acids with perfect complementarity andnucleic acids with mismatches.

In alternative embodiments, neither redox site has a long lived excitedstate, and electron transfer measurements depend upon bimoleculargeneration of a kinetic intermediate. For a review, see Winkler et al.,supra. This intermediate then relaxes to the thermodynamic product viaintramolecular electron transfer using a quencher, as seen below:

D-A+hv→D-A*

D-A*+Q→D-A⁺+Q⁻

D-A⁺→D⁺-A

D⁺-A+Q⁻→D-A+Q

The upper limit of measurable intramolecular electron transfer ratesusing this method is about 10⁴ per second.

Alternative embodiments use the pulse-radiolylic generation of reducingor oxidizing radicals, which inject electrons into a donor or removeelectrons from a donor, as reviewed in Winkler et al., supra.

As is appreciated in the art, there are a variety of ways to initiateand detect the electron transfer.

Electron transfer can be initiated and detected using a wide variety ofmethods, including, but not limited to, electrical, electrochemical,electromagnetic radiation (optical) and chemical methods. It is possibleto make a variety of compositions utilizing different electron transfermoieties depending on the desired methods of initiating electrontransfer and detection of electron transfer. Table 2 depicts a varietyof preferred combinations for initiation and detection of electrontransfer in the complexes of the invention.

TABLE 2 Initiation Detection Description light light absorbance,fluorescence, phosphorescence, refractive index, surface plasmonresonance, electron spin resonance light current amperommetry,voltammetry, capacitance, impedence, opto-electronic detection, photo-amperometry light plus light absorbance, fluorescence, phosphorescence,electronic refractive index, surface plasmon resonance, initiationelectron spin resonance light plus current amperommetry, voltammetry,capacitance, electronic impedence, opto-electronic detection, photo-initiation amperommetry, amperommetric detection, cyclic voltammetryelectronic current amperommetry, voltammetry, capacitance, initiationimpedence, amperommetric detection, cyclic voltammetry electronic lightchemiluminescence, initiation electrochemiluminescence,electroluminescence

By “light” herein is meant electromagnetic radiation, with light in theUV, visible and infrared range being preferred, and UV and visible beingthe most preferred.

In a preferred embodiment, initiation of electron transfer is via director indirect photoactivation (“light in”). Simply, electromagneticradiation of appropriate wavelength strikes the redox molecule on oneend of the DNA causing excitation of a donor moiety electron whicheither decays immediately or is involved in intramolecular electrontransfer. The efficiency with which electron transfer is induced dependsupon the electronic coupling between the electron donor and acceptor andtherefore depends on whether the nucleic acid is single or doublestranded. In addition, the efficiency of electron transfer depends uponthe extinction coefficient of the electron donor at the wavelength oflight used (higher is better) and upon the lifetime of the donorelectron excited state (longer is better). Preferred donor complexestherefore include acridine orange, N,N′-dimethyl-2,7-diazapyreniumdichloride (DAP²⁺), methylviologen, ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def:6,5,10-d′e′f′)diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride]. Transition metal donors and acceptors include complexesof ruthenium, rhenium and osmium (most preferred) where at least one ofthe ligands is a chromophore.

Photoactivation can also be used to excite “mediators” that transferenergy to the electron donor moiety on the DNA via an inter-molecularprocess. Such mediators include water soluble and stable complexes ofthe transition metals, including molybdenum and tungsten halides,trisbipyridyl complexes of rhenium, osmium and ruthenium. In addition,other examples include bipyridyl and pyridyl complexes such asRe(bpy)(CO)₃X where X is a halide and Re(py)₄O₂. Other examples includetransition metal dimers such as [Re₂Cl₈]²⁻ and [Pt₂(P₂O₅H₂)₄]⁴⁻.Ruthenium trisbypyridine (Ru²⁺(bpy)₃) is most preferred.

In the preferred embodiment, electron transfer occurs afterphotoinduction with a laser. In this embodiment, electron donor moietiesmay, after donating an electron, serve as electron acceptors undercertain circumstances. Similarly, electron acceptor moieties may serveas electron donors under certain circumstances.

A preferred embodiment utilizes electronic activation, with voltagebeing preferred. A potential is applied to a sample containing modifiednucleic acid probes either via a direct linkage of the modified nucleicacid to an electrode, or using electron transport mediators. Directlinkage can involve a redox active polymer to shuttle electrons from(and to, if the electrode is also used for detection) the electrode.Such polymers are outlined below. Alternatively, the direct connectioncan involve a relatively poorly conducting linkage provided the linkageis kept reasonably short (less than six sigma bonds). Preferred linkageswill be three or fewer sigma bonds in length to allow efficient transferof electrons from the electrode, as is outlined below.

Indirect electron transfer initiation involves electron transfermediators or effective diffusional electron donors and acceptors such aswater soluble ferrocene/ferricinium, hydroquinones/quinones, reducibleand oxidizable components of organic salts, cobaltocenes, the hexa- andoctacyanides of molybdenum, tungsten and iron. In addition, otherexamples include macrocycles and chelating ligands of transition metalssuch as cobalt, ruthenium and nickel, including Co(ethylenediamine)₃ andRu(ethylenediamine)₃ and the trisbypyridyl and hexamine complexes oftransition metals such as Co, Ru, Fe, and Os. See K. Alyanasundaram,Coord. Chem. Rev. V.46, p. 159, 1982. Finally, organic molecules such as4,4′-bipyridine and 4-mercaptopyridine are examples where ferrocene ismost preferred.

Precise control and variations in the applied potential can be via apotentiostat and a three electrode system (one reference, one sample andone counter electrode). This allows matching of applied potential topeak electron transfer potential of the system which depends in part onthe choice of electron acceptors attached to the nucleic acid. Highdriving forces are achieved using bisbipyridyl complexes of transitionmetals, for example, ruthenium and rhenium bisbipyridyl complexes suchas (Ru(bpy)₂im-) as electron acceptors.

Alternatively, electrochemical initiation of electron transfer may beused. The redox states of the electron donating and accepting moietiesattached to nucleic acid can be electrochemicaly changed using watersoluble chemical oxidants and reductants, either with or without photo-or electrical activation. Such compounds include numerous derivativesknown in the art (T. Kuwana, Electrochemical Studies of BiologicalSystems, (D. T. Sawyer Ed.) ACS Symp. Series #38, (1977)) and includehexacyano iron complexes, zinc-mercury amalgam, and trisphenanthrolinecomplexes of ruthenium and iron.

Electron transfer through nucleic acid can be detected in a variety ofways. A variety of detection methods may be used, including, but notlimited to, optical detection, which includes fluorescence,phosphorescence, and refractive index; and electronic detection,including, but not limited to, amperommetry, voltammetry, capacitanceand impedence. These methods include time or frequency dependent methodsbased on AC or DC currents, pulsed methods, lock-in techniques,filtering (high pass, low pass, band pass), and time-resolved techniquesincluding time-resolved fluorescence. In some embodiments, all that isrequired is electron transfer detection; in others, the rate of electrontransfer may be determined.

In one embodiment, the efficient transfer of electrons from one end of anucleic acid double helix to the other results in stereotyped changes inthe redox state of both the electron donor and acceptor. With manyelectron transfer moieties including the complexes of rutheniumcontaining bipyridine, pyridine and imidazole rings, these changes inredox state are associated with changes in spectral properties (“lightout”). Significant differences in absorbance are observed betweenreduced and oxidized states for these molecules. These differences canbe monitored using a spectrophotometer or simple photomultiplier tubedevice.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction (large extinctioncoefficient “deltas”) resulting in highly sensitive monitoring ofelectron transfer. Such examples include Ru(NH₃)₄py and Ru(bpy)₂im aspreferred examples. It should be understood that only the donor oracceptor that is being monitored by absorbance need have ideal spectralcharacteristics. That is, the electron acceptor can be opticallyinvisible if only the electron donor is monitored for absorbancechanges.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence. Highly efficient electron transfer through double strandednucleic acid can, for example, result in the production of fluorescentRu(4,7-biphenyl₂-phenanthroline)₃ ²⁺ at one end of a nucleic acid probewhen the electron transfer moiety on the other end is excited. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic sensorswith nucleic acid probes in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the Fluorlmagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺ andRu(4,4′-diphenyl-2,2′-bipyridine)₃ ²⁺.

Alternatively, a reduction in fluorescence associated with hybridizationcan be measured using these systems. An electron transfer “donor”molecule that fluoresces readily when on single stranded nucleic acid(with an “acceptor” on the other end) will undergo a reduction influorescent intensity when complementary nucleic acid binds the probeallowing efficient transfer of the excited state electron. This drop influorescence can be easily monitored as an indicator of the presence ofa target sequence using the same methods as those above.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some electron transfer moietiessuch as Ru²⁺(bpy)₃, direct luminescence accompanies excited state decay.Changes in this property are associated with nucleic acid hybridizationand can be monitored with a simple photomultiplier tube arrangement (seeBlackburn, G. F. Clin. Chem. 37: 1534-1539 (1991); and Juris et al.,supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer through nucleicacid is via amperometric detection, either directly using a covalentlyattached electrode, or indirectly using electron transport “mediators”to shuttle electrons from the nucleic acid to an electrode. Modes ofattaching nucleic acids to electrodes and possible mediators aredescribed below. An amperometric detector would resemble the numerousenzyme-based biosensors currently used to monitor blood glucose, forexample. This method of detection involves applying a potential (ascompared to a separate reference electrode) between the nucleicacid-conjugated electrode and an auxiliary (counter) electrode in thesample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target nucleic acid; that is, the single stranded probe exhibits adifferent rate than the probe hybridized to the target sequence. Thediffering efficiencies of electron transfer result in differing currentsbeing generated in the electrode.

The device for measuring electron transfer amperometrically involvessensitive (nanoamp to picoamp) current detection and includes a means ofcontrolling the voltage potential, usually a potentiostat. This voltageis optimized with reference to the potential of the electron donatingcomplex on the nucleic acid. Possible electron donating complexesinclude those previously mentioned with complexes of ruthenium beingpreferred and complexes of rhenium being most preferred.

In a preferred embodiment, alternative electron detection modes areutilizes. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer through nucleic acid. In addition, otherproperties of insulators (such as resistance) and of conductors (such asconductivity, impedance and capacitance) could be used to monitorelectron transfer through nucleic acid. Finally, any system thatgenerates a current (such as electron transfer) also generates a smallmagnetic field, which may be monitored in some embodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, between two and four orders of magnitudeimprovements in signal-to-noise may be achieved. This is particularlytrue using AC methodology, as is more fully described below.

In a preferred embodiment, electron transfer is initiated and detectedusing alternating current (AC) methods, particularly when one of theelectron transfer moieties is an electrode; that is, when the nucleicacid is attached to an electrode. This system is particularlyadvantageous for a number of reasons. In general, the use of ACtechniques can result in good signals and low background noise. Withoutbeing bound by theory, there are a number of possible contributors tobackground noise, or “parasitic” signals, i.e. detectable signals thatare inherent to the system but are not the result of the presence of thetarget sequence.

However, all of the contributors to parasitic noise will generally givevery fast signals; that is, the rate of electron transfer through thedouble helix, i.e. the “π-way”, is generally significantly slower thanthe rate of electron transfer of the parasitic components, such as thecontribution of charge carriers in solution, and other “shortcircuiting” mechanisms. As a result, the parasitic components aregenerally all phase related; that is, they exhibit a constant phasedelay or phase shift that will scale directly with frequency. Thehybridization complex, in contrast, exhibits a time delay between theinput and output signals, which is independent of frequency. Thus, forthe hybridization signal, the time it takes electrons to travel betweenthe electron transfer moieties will remain constant and relatively largeas compared to parasitic background. As a consequence, at differentfrequencies, the phase of the system will change. This is very similarto the time domain detection used in fluorescent systems.

This difference can be exploited in various methods to decrease thesignal to noise ratio. Accordingly, the preferred detection methodscomprise applying an AC input signal to a hybridization complexcomprising a first single stranded nucleic acid containing an electrodeand a second electron transfer moiety and a target single strandednucleic acid. The presence of the target nucleic acid (hybridizationcomplex) is detected via an output signal characteristic of electrontransfer through the hybridization complex; that is, the output signalis characteristic of the hybridization complex rather than the parasiticcomponents or single stranded nucleic acid. Thus, for example, theoutput signal will exhibit a time delay dependent on the rate ofelectron transfer between the two electron transfer moieties. It shouldbe noted that this time delay will also vary depending on the distancebetween the electron transfer moieties; the farther apart the electrontransfer moieties are, the longer the time delay.

In a preferred embodiment, the input signals are applied at a pluralityof frequencies, since this again allows the distinction between truesignal and noise. “Plurality” in this context means at least two, andpreferably more, frequencies. In general, the AC frequencies will rangefrom about 0.1 Hz to about 10 mHz, with from about 1 Hz to 100 KHz beingpreferred.

In a preferred embodiment, DNA is modified by the addition of electrondonor and electron acceptor moieties. In an alternative embodiment, RNAis modified. In a further embodiment, a double stranded nucleic acid foruse as a bioconductor will contain some deoxyribose nucleosides, someribose nucleosides, and a mixture of adenosine, thymidine, cytosine,guanine and uracil bases.

In accordance with a further aspect of the invention, the preferredformulations for donors and acceptors will possess a transition metalcovalently attached to a series of ligands and further covalentlyattached to an amine group as part of the ribose ring (2′ or 3′position) or to a nitrogen or sulfur atom as part of a nucleoside dimerlinked by a peptide bond, phosphoramidate bond, phosphorothioate bond,phosphorodithioate bond or O-methyl phosphoramidate bond.

In a preferred embodiment, an oligonucleotide containing at least oneelectron transfer moiety is attached to an electrode, which also servesas an electron transfer moiety, thus forming a single stranded nucleicacid with both an electron donor moiety and an electron acceptor moietyattached in the manner outlined above. Preferably, the single strandednucleic acid containing an electron transfer moiety is attachedcovalently or in such a way that allows the transfer of electrons fromthe electrode to the single stranded nucleic acid in order to allowelectron transfer between the electron donor and acceptor. Preferably,the non-electrode electron transfer moiety is attached at or near theterminus of the oligonucleotide, such that the probe sequence to behybridized to the target sequence is between the donor and acceptor. Theelectrode may be immersed in a sample containing the target sequencesuch that the target sequence hybridizes to the probe and electrontransfer may be detected using the techniques outlined above.

In an additional embodiment, two nucleic acids are utilized as probes asdescribed previously. For example, one nucleic acid is covalentlyattached to a solid electrode which serves as an electron transfermoiety, and the other, with a covalently attached electron transfermoiety, is free in solution. Upon hybridization of a target sequence,the two nucleic acids are aligned such that electron transfer betweenthe electron transfer moiety of the hybridized nucleic acid and theelectrode occurs. The electron transfer is detected as outlined above,using techniques well known in the art.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.The references cited herein are expressly incorporated by reference.

EXAMPLES

The amino-modified monomer units are prepared by variation of publishedprocedures and are incorporated into a growing oligonucleotide bystandard synthetic techniques. The procedure is applicable to both DNAand RNA derivatives.

Example 1 Synthesis of an Oligonucleotide Duplex with Electron TransferMoieties at the 5′ Termini

In this example an eight nucleotide double stranded nucleic acid wasproduced, with each single strand having a single electron transfermoiety covalently attached to the 5′ terminal uridine nucleotide at the2′ carbon of the ribose sugar.

Step 1: Synthesis of 5′-di(p-methoxyphenyl)methylether-2′-(trifluoroacetamido)-2′-deoxyuridine

2′-(trifluoroacetamido)-2′-deoxyuridine (2.0 g, 5.9 mmoles) prepared byminor modification of published procedures (Imazawa, supra) wasrepeatedly dissolved in a minimum of very dry CH₃CN and rotaryevaporated to dryness and then transferred to inert atmosphere vacuumline and further dried for a period of 1 hour. The following procedurefor the synthesis of the material was adapted from Gait (supra): Underpositive pressure argon, the material was dissolved in freshly dried anddistilled pyridine and with stirring, 0.05 equivalents (wt.) of4-dimethylaminopyridine (DMAP), 1.5 equivalents of triethylamine (TEA)and 1.2 equivalents of 4,4′-dimethoxytrityl chloride (DMTr-Cl) wereadded to the reaction mixture. The progress of the reaction wasmonitored by silica gel TLC (98:2 methylene chloride:methanol, mobilephase). After 30 minutes, an additional 0.5 equivalents each of DMTr-Cland TEA were added and the reaction allowed to proceed for an additionalthree hours. To this reaction mixture was added an equal volume of waterand the solution extracted several times with diethyl ether. The etherlayers were rotary evaporated to dryness, redissolved in a minimumamount of methylene chloride and purified by flash chromatography (99:1methylene chloride:methanol, mobile phase), to obtain the5′-di(p-methoxyphenyl)methylether-2′-(trifluoroacetamido)-2′-deoxyuridine product.

Step 2: 5′-2′-aminouridine-GCTACGA and 5′-2′-aminouridine-CGTAGCA

5′-di(p-methoxyphenyl)methylether-2′-(trifluoroacetamido)-2′-deoxyuridine was dried under reducedpressure (glass) and dissolved in freshly dried and distilled CH₃CN andplaced in a specially made conical vial and placed on an ABl DNAsynthesizer. The program for the preparation of standard (i.e.unmodified) oligonucleotides was altered during the final base(amino-modified) addition to a 15-30 minute coupling time. Theoligonucleotide was cleaved from the column by standard procedures andpurified by C-18 reverse phase HPLC. In this manner5′-2′-aminouridine-GCTACGA and 5′-2′-aminouridine-CGTAGCA were prepared.In addition, unmodified complementary strands to both products were madefor use in the electron transfer moiety synthesis below.

Step 3: 5′-2′-ruthenium bisbipyridineimidazole-aminouridine-GCTACGA

5′-2′-aminouridine GCTACGA produced in the previous step was annealed tothe complementary unmodified strand using standard techniques. Allmanipulations of the annealed duplex, prior to the addition of thetransition metal complex were handled at 4° C. In order to insure thatthe DNA remained annealed during modification, the reactions wereperformed in 1M salt. The 5′-amino modified duplex DNA was dissolved in0.2 M HEPES, 0.8 M NaCl, pH 6.8 and repeatedly evacuated on a Schlenkline. Previously prepared ruthenium bisbipyridine carbonate wasdissolved in the above buffer and oxygen was removed by repeatedevacuation and purging with argon via a Schlenk line. The rutheniumcomplex was transferred to the DNA solution via cannulation(argon/vacuum) and the reaction allowed to proceed under positivepressure argon with stirring for 24 hours. To this reaction, 50equivalents of imidazole was added to the flask and the reaction allowedto proceed for an additional 24 hours. The reaction mixture was removedfrom the vacuum line and applied to a PD-10 gel filtration column andeluted with water to remove excess ruthenium complex. The volume of thecollected fractions was reduced to dryness via a speed vac and the solidtaken up in 0.1 M triethylammonium acetate (TEAC) pH 6.0. The duplex DNAwas heated to 60° C. for 15 minutes with 50% formamide to denature theduplex. The single stranded DNA was purified using a C-18 reverse phaseHPLC column equipped with a diode array detector and employing agradient from 3% to 35% acetonitrile in 0.1 M TEAC, pH 6.0.

Step 4: 5′-2′-ruthenium tetraminepyridine-aminouridine-CGTAGCA

5′-aminouridine-CGTAGCA (0.3 μm) was dissolved in 0.2 M HEPES, 0.8 MNaCl buffer, pH 6.8 and degassed on the vacuum line. To a 10 ml conicalshaped flask equipped with a stirring bar and septum was slurriedRu(III) tetraaminepyridine chloride (10 μm), in the same buffer. In aseparate flask, Zn/Hg amalgam was prepared and dried under reducedpressure and the ruthenium(III) solution transferred (via cannulation)to the Zn/Hg amalgam. The immediate formation of a clear yellow solution(λ_(max)=406 nm) indicated that the reduced form of the ruthenium hadbeen achieved and the reaction allowed to proceed for 30 minutes. Thissolution was transferred to the flask containing the amino-modified DNAand the reaction allowed to proceed at room temperature for 24 hoursunder argon. The reaction mixture was removed from the vacuum line and a50 fold excess of cobalt EDTA (Kirschner, Inorganic Synthesis (1957), pp186) added to the solution. The solution was applied to Sephadex G-25gel filtration column to remove excess ruthenium complex and furtherpurified by reverse phase HPLC as described above. The two rutheniummodified nucleotides were annealed by standard techniques andcharacterized (see Example 5).

Example 2 Synthesis of Long DNA Duplexes with Electron Transfer Moietiesat the 5′ Termini

In this example, an in vitro DNA amplification technique, PCR (reviewedin Abramson et al., Curr. Op. in Biotech. 4:41-47 (1993)) is used togenerate modified duplex DNA by polymerization of nucleotides offmodified primer strands (Saiki et al., Science 239:487 (1988)). Twooligonucleotides 18 bases in length and not complementary to each otherare synthesized with amino-modification to the 2′-ribose position of the5′ nucleotides, as in example 1.

A series of oligonucleotides of increasing lengths starting at 40 basesare chemically synthesized using standard chemistry. Each of the PCRtemplates shares a 5′ sequence identical to one modified 18 mer. The 3′end of the template oligonucleotide shares a sequence complementary tothe other 18 mer.

PCR rapidly generates modified duplex DNA by the catalysis of 5′-3′ DNAsynthesis off of each of the modified 18 mers using the unmodifiedstrand as a template. One hundred nanomoles of each of the two modified18 mers are mixed in 1 ml of an aqueous solution containing 2,000 unitsof Taq polymerase, deoxyribonucleoside triphosphates at 0.2 M each, 50mM KCl, 10 mM Tris-Cl, pH 8.8, 1.5 mM MgCl₂, 3 mM dithiothreitol and 0.1mg/ml bovine serum albumin. One femtomole of the template strand 40bases in length is added to the mixture. The sample is heated at 94° C.for one minute for denaturation, two minutes at 55° C. for annealing andthree minutes at 72° C. for extension. This cycle is repeated 30 timesusing an automated thermal cycler.

The amplified template sequences with transition metal complexes on both5′ termini are purified by agarose gel electrophoresis and used directlyin electron transfer applications.

Example 3 Synthesis of Covalently Bound Electron Transfer Moieties atInternucleotide Linkages of Duplex DNA

In this example, alternative backbones to phophodiester linkages ofoligonucleotides are employed. Functional groups incorporated into theseinternucleotide linkages serve as the site for covalent attachment ofthe electron transfer moieties. These alternate internucleotide linkagesinclude, but are not limited to, peptide bonds, phosphoramidate bonds,phosphorothioate bonds, phosphorodithioate bonds andO-methylphosphoramidate bonds.

The preparation of peptide nucleic acid (PNA) follows literatureprocedures (See Engholm, supra), with the synthesis of Boc-protectedpentaflurophenyl ester of the chosen base (thymidine). The resulting PNAmay be prepared employing Merrifield's solid-phase approach (Merrifield,Science, 232:341 (1986)), using a single coupling protocol with 0.1 M ofthe thiminyl monomer in 30% (v/v) DMF in CH₂Cl₂. The progress of thereaction is followed by quantiative ninhydrin analysis (Sarin, Anal.Biochem., 117:147 (1981)). The resulting PNA may be modified with anappropriate transition metal complex as outlined in example 1.

The synthesis of phosphoramidate (Beaucage, supra, Letsinger, supra,Sawai, supra) and N-alkylphosphoramidates (Jager, supra) internucleotidelinkages follows standard literature procedures with only slightmodification (the procedures are halted after the addition of a singlebase to the solid support and then cleaved to obtain a dinucleotidephosphoramidate). A typical example is the preparation of the phenylester of5′O-isobutyloxycarbonylthymidyl-(3′-5′)-5′-amino-5′-deoxythymidine(Letsinger, J. Org. Chem., supra). The dimer units are substituted forstandard oligonucleotides at chosen intervals during the preparation ofDNA using established automated techniques. Transition metalmodification of the modified linkages takes place as described inExample 1.

The synthesis of phosphorothioate and phosphorodithioate (Eckstein,supra, and references within) internucleotide linkages is welldocumented. A published protocol utilizes an Applied Biosystems DNAsynthesizer using a modified β-cyanoethylphosphoramidite cycle that capsafter sulphurization with tetraethylthiuram disulfide (TETID) (lyer, J.Org. Chem. 55:4693 (1990)). The phosphorothioate and phosphorodithioateanalogs are prepared as dimers and cleaved from the solid support andpurified by HPLC (acetonitrile/triethylammonium acetate mobile phase).

Example 4 Synthesis of Two Oligonucleotides Each with an ElectronTransfer Moiety at the 5′ Terminus

In this example, two oligonucleotides are made which hybridize to asingle target sequence, without intervening sequences. Oneoligonucleotide has an electron donor moiety covalently attached to the5′ terminus, and the other has an electron acceptor moiety covalentlyattached to the 5′ terminus. In this example, the electron transferspecies are attached via a uradine nucleotide, but one skilled in theart will understand the present methods can be used to modify any of thenucleotides. In addition, one skilled in the art will recognize that theprocedure is not limited to the generation of 8-mers, but is useful inthe generation of oligonucleotide probes of varying lengths.

The procedure is exactly as in Example 1, except that the 8-mersgenerated are not complementary to each other, and instead arecomplementary to a target sequence of 16 nucleotides. Thus the finalannealing step of step 4 of Example 1 is not done. Instead, the twomodified oligonucleotides are annealed to the target sequence, and theresulting complex is characterized as in Example 5.

Example 5 Characterization of Modified Nucleic Acids

Enzymatic Digestion

The modified oligonucleotides of example 1 were subjected to enzymaticdigestion using established protocols and converted to their constituentnucleosides by sequential reaction with phosphodiesterase and alkalinephosphatase. By comparison of the experimentally obtained integratedHPLC profiles and UV-vis spectra of the digested oligonucleotides tostandards (including 2′-aminouridine and 2′-aminoadenine), the presenceof the amino-modified base at the predicted retention time andcharacteristic UV-vis spectra was confirmed. An identical procedure wascarried out on the transition metal modified duplex DNA and assignmentsof constituent nucleosides demonstrated single-site modification at thepredicted site.

Fluorescent Labeled Amino-Modified Oligonucleotides

It has been demonstrated that the fluorochrome, fluoresceinisothiocyanate (FITC) is specific for labeling primary amines onmodified oligonucleotides while not bonding to amines or amides presenton nucleotide bases (Haugland, Handbook of Fluorescent Probes andResearch Chemicals, 5th Edition, (1992)). This reaction was carried outusing the amino-oligonucleotide synthesized as described in example 1and on an identical bases sequence without the 2′-amino-ribose grouppresent. Fluorescence spectroscopic measurements were acquired on boththese oligonucleotides and the results confirm the presence of the amineon the 5′-terminal ribose ring.

Thermodynamic Melting Curves of Modified Duplex DNA

A well established technique for measuring thermodynamic parameters ofduplex DNA is the acquisition of DNA melting curves. A series of meltingcurves as a function of concentration of the modified duplex DNA wasmeasured via temperature controlled UV-vis (Hewlett-Packard), usingtechniques well known in the art. These results confirm thathybridization of the amino-modified and transition metal modified DNAhad taken place. In addition, the results indicate that the modified DNAform a stable duplex comparable to the stability of unmodifiedoligonucleotide standards.

Two Dimensional Nuclear Magnetic Resonance (NMR) Spectroscopy

The amino-modified oligonucleotides synthesized as a part of this workwere prepared in sufficient quantities (6 micromoles) to permit theassignment of the ′H proton NMR spectra using a 600 MHz Varian NMRspectrometer.

Measurement of the Rate of Electron Transfer

An excellent review of the measurement techniques is found in Winkler etal., Chem. Rev. 92:369-379 (1992). The donor is Ru(bpy)₂(NHuridine)im,E⁰˜1 V, and the acceptor is Ru(NH₃)₄py(NHuridine)im, E⁰˜330 mV. Thepurified transition metal modified oligonucleotides(U_(NHRu(bpy)2im)GCATCGA and U_(NHRu(NH3)4(py)im)CGATGCA were annealedby heating an equal molar mixture of the oligonucleotides (30 μmolar: 60nmoles of DNA in 2 ml buffer) in pH 6.8 (100 mM NaPi, 900 mM NaCl) to60° C. for 10 minutes and slowly cooling to room temperature over aperiod of 4 hours. The solution was transferred to an inert atmospherecuvette equipped with adapters for attachment to a vacuum line and amagnetic stirring bar. The solution was degassed several times and thesealed apparatus refilled repeatedly with Ar gas.

The entire apparatus was inserted into a cuvette holder as part of theset-up using the XeCl excimer-pumped dye laser and data acquired atseveral wavelengths including 360, 410, 460 and 480 nm. The photoinducedelectron transfer rate is 1.6×10⁶ s⁻¹ over a distance of 28 Å.

Example 6 Synthesis of a Single Stranded Nucleic Acid Labeled with TwoElectron Transfer Moieties

This example uses the basic procedures described earlier to generate twomodified oligonucleotides each with an electron transfer moietyattached. Ligation of the two modified strands to each other produces adoubly labeled nucleic acid with any of four configurations: 5′ and 3′labeled termini, 5′ labeled terminus and internal nucleotide label, 3′labeled terminus and internal nucleotide label, and double internalnucleotide labels. Specifically, the synthesis of an oligonucleotide 24bases in length with an electron transfer donor moiety on the 5′ end andan internal electron transfer moiety is described.

Five hundred nanomoles of each of two 5′-labeled oligonucleotides 12bases in length are synthesized as detailed above with ruthenium (II)bisbipyridine imidazole on one oligonucleotide, “D” and ruthenium (III)tetraamine pyridine on a second oligonucleotide, “A”.

An unmodified oligonucleotide 24 bases in length and complementary tothe juxtaposition of oligonucleotide “D” followed in the 5′ to 3′direction by oligonucleotide “A” is produced by standard synthetictechniques. Five hundred nanomoles of this hybridization template isadded to a mixture of oligonucleotides “A” and “D” in 5 ml of an aqueoussolution containing 500 mM Tris-Cl, pH 7.5, 50 mM MgCl₂, 50 mMdithiothreitol and 5 mg/ml gelatin. To promote maximal hybridization oflabeled oligonucleotides to the complementary strand, the mixture isincubated at 60° C. for 10 minutes then cooled slowly at a rate ofapproximately 10° C. per hour to a final temperature of 12° C. Theenzymatic ligation of the two labeled strands is achieved with T4 DNAligase at 12° C. to prevent the ligation and oligomerization of theduplexed DNA to other duplexes (blunt end ligation). Alternatively, E.coli DNA ligase can be used as it does not catalyze blunt end ligation.

One hundred Weiss units of T4 DNA ligase is added to the annealed DNAand adenosine triphosphate is added to a final concentration of 0.5 mM.The reaction which catalyzes the formation of a phosphodiester linkagebetween the 5′ terminal phosphate of oligonucleotide “A” and the 3′terminal hydroxyl group of oligonucleotide “D” is allowed to proceed for18 hours at 12° C. The reaction is terminated by heat inactivation ofthe enzyme at 75° C. for 10 minutes. The doubly labeled oligonucleotideis separated from the singly labeled oligonucleotides and thecomplementary unlabeled oligonucleotide by HPLC in the presence of ureaas in the previous examples. The doubly labeled oligonucleotide of thisexample is ideally suited for use as a photoactive gene probe asdetailed below.

Example 7 Use of a Doubly Modified Oligonucleotide with ElectronTransfer Moieties as a Photoactive Probe for Homologous Nucleic AcidSequence Detection

This example utilizes the oligonucleotide 24 mer of example 6 in aunique type of gene-probe assay in which removal of unhybridized probeprior to signal detection is not required. In the assay procedure, aregion of the gag gene of human immunodeficiency virus type I (HIV-I) isamplified by the polymerase chain reaction (Saiki et al., Science239:487-491 (1988)). This region of HIV-I is highly conserved amongdifferent clinical isolates.

The amplified target DNA versus controls lacking in HIV-I DNA are addedto a hybridization solution of 6×SSC (0.9 M NaCl, 0.09 M Na citrate, pH7.2) containing 50 nanomoles of doubly labeled 24 mer probe of example6. Hybridization is allowed to proceed at 60° C. for 10 minutes withgentle agitation. Detection of electron transfer following laserexcitation is carried out as in example 5. Control samples which lackthe hybridized probe show negligible electron transfer rates. Probeshybridized to the gag sequence show efficient and rapid electrontransfer through the DNA double helix, providing a highly specific,homogeneous and automatable HIV-I detection assay.

A similar homogeneous gene probe assay involves the use of two probes,one an electron donor and the other an electron acceptor, whichhybridize with the gag region of HIV-I in a tandem configuration, oneprobe abutting the other. In this assay, electronic coupling between thetwo electron transfer moieties depends entirely on hybridization withthe target DNA. If appropriate, the electron transfer from one probe tothe other is enhanced by the ligation of the juxtaposed ends using T4DNA ligase as in example 6.

Example 8 Preparation of a Hydroxythiol for Attachment to a GoldElectrode

OH(CH₂)₁₆OH was purchased from Aldrich and the monoacetate form preparedby slurring the material in dry CH₂Cl₂. 0.5 equiv. ofdimethylaminopyridine was added along with 1.4 equivalents oftriethylamine and I equivalent of acetic anhydride. The reaction wasallowed to proceed for 2 hours and purified by flash chromatography(80:20 hexane:diethyl ether.

The monoacetate compound was converted to the monotosylate-monoacetateusing p-TSOCI by literature procedures and then treated with triphenylmethylmercaptan. To remove the monoacetate, the product was dissolved inMeOH (1 mmol, 9 ml), cooled to 0° C., and aqueous solution of NaOH (1mmol, in 2 ml water) added. The temperature was allowed to rise to roomtemperature slowly, and the reaction followed by TLC (5% MeOH/CH₂Cl₂).When the ester was gone the mixture was recooled to 0° C., and acidifiedwith KHSO₄ to pH 5-6 using pH paper. The MeOH was evaporated, and theresidue was extracted with CH₂Cl₂ (200 ml), dried (Na₂SO₄), evaporatedand checked via TLC. The material was phosphoroamidited by standardprocedures. This material was inserted into the DNA synthesizer and anmodified oligonucleotide produced. The phosphoramidited oligonucleotidewas modified with a ruthenium complex by adding Ru(bpy)₂CO₃ followed byimidazole to yield a Ru(bpy)₂im oligonucleotide. The trityl protectinggroup was removed by dissolving the nucleotide in 200 μl of 0.1 Mtriethylammonium acetate (TEAA) buffer, pH 7.5. 30 μl of 1 M silvernitrate solution was added and the mixture vortexed and incubated atroom temperature for 30 minutes. 50μ of 1 M dithiothritol (DTT) wasadded, the mixture vortexed and incubated for 15 minutes, at which pointit was microcentrifuged for 15 minutes to remove precipitated Ag+DTT.The supernatant was collected and the pellet was washed with 100 μl ofTEAA buffer and the solutions pooled. The resulting oligonucleotide wasthen attached to the gold surface by standard techniques.

Example 9 Synthesis of a Single Stranded Nucleic Acid Containing Both anElectron Acceptor and an Electron Donor Moiety

In order to evaluate the path dependent nature of the electron transferprocess through duplex DNA, an oligonucleotide was prepared with anelectron donor at the 3′ end and an electron acceptor at the 5′ end.This multiply-modified oligonucleotide was prepared by synthesizing aderivative with an amine at the 2′-position of the terminal ribose ofboth ends.

Synthesis of Bis-3′,5′-2′-deoxyuridine Oligonucleotides

A DMT-2′-N-trifluoroacetyl-protected phosphoroamidite of2′-amino-2′-deoxyuridine (U_(NH2)) was prepared as described earlier andreacted with succinic anhydride. This material was reacted withp-nitrophenol to produce the precursor for the attachment to thecontroller pore glass (GPG) resin as in FIG. 6A. The modifiedoligonucleotide were assembled by standard solid phase automated DNAsynthesis techniques and the bis-3′,5′,-2′-amino-2′-deoxyuridineoligonucleotide isolated and characterized by mass spectrometry andHPL(, digestion analysis. In addition, the aminoribose oligomers andtheir complements were reacted with FITC under conditions that favorlabeling of primary amines. As expected, only the 2′-amino-2′deoxyribosesite was labeled verifying the presence of a primary amine on the DNA.As an example, a 11 base pair sequence was prepared (calc. forU_(NH2)CTCCTACACU_(NH2)-3229; found 3229.1) and the subsequent digestionmap was consistent with the proposed structure. The metal modificationof the bis-amino modified oligonucleotide was performed in a similarmanner. The new metal-modified oligonucleotides were characterized byfluorescent labelling, enzymatic digestion, and duplex-meltingtemperature studies.

Thermal denaturing and annealing experiments display similar meltingtemperatures for both ruthenium and aminoribose oligomers. In addition,the amino-modified duplex DNA has been characterized by 2D NMR. Thesedata confirm that the donors and acceptors are covalently attached tothe 2′-amino-2′deoxyribose position and indicate that the DNA structureis unperturbed by the presence of the ruthenium complexes.

We claim:
 1. A method of detecting the presence of a sequence in anucleic acid sample, said method comprising: a) applying an input signalto an electrode comprising a hybridization complex, wherein saidhybridization complex comprises: 1) a single stranded nucleic acid; 2) atarget sequence hybridized to said single stranded nucleic acid; and 3)at least one covalently attached electron donor moiety; such that anelectron is transferred from said electron donor moiety to saidelectrode such that the redox state of said electron donor moiety ischanged to form an electron acceptor moiety; b) transferring an electronfrom a reductant to said electron acceptor moiety; and c) detectingelectron transfer between said electron donor moiety and said electrode,wherein the presence of electron transfer is an indication of thepresence of said target sequence.
 2. A method according to claim 1wherein said detecting is done by detecting an output signalcharacteristic of electron transfer between said donor moiety and saidelectrode.
 3. A method of detecting the presence of a sequence in anucleic acid sample, said method comprising: a) applying an input signalto an electrode comprising a hybridization complex, wherein saidhybridization complex comprises: 1) a single stranded nucleic acid; 2) atarget sequence hybridized to said single stranded nucleic acid; and 3)at least one covalently attached electron acceptor moiety; such that anelectron is transferred from said electron acceptor moiety to saidelectrode such that the redox state of said electron acceptor moiety ischanged to form an electron donor moiety; b) transferring an electronfrom an oxidant to said electron donor moiety; and c) detecting electrontransfer between said electron acceptor moiety and said electrode,wherein the presence of electron transfer is an indication of thepresence of said target sequence.
 4. A method according to claim 3wherein said detecting is done by detecting an output signalcharacteristic of electron transfer between said acceptor moiety andsaid electrode.
 5. A method according to claim 2 or 4 wherein saidoutput signal is a current.
 6. A method according to claim 1 or 3wherein said single stranded nucleic acid comprises said electrode.
 7. Amethod according to claim 1 or 3 wherein said single stranded nucleicacid comprises said electron donor moiety.
 8. A method according toclaim 1 or 3 wherein said target sequence comprises said electron donormoiety.
 9. A method according to claim 1 or 3 wherein said hybridizationcomplex comprises a plurality of electron donor moieties.
 10. A methodaccording to claim 1 or 3 wherein said electron donor moiety is atransition metal complex.
 11. A method according to claim 10 whereinsaid transition metal complex comprises a metal selected from the groupconsisting of ruthenium, rhenium, osmium, platinum, copper and iron. 12.A method according to claim 1 or 3 wherein said electron donor moiety isan organic electron donor moiety.
 13. A method according to claim 1wherein said reductant is a hexacyano iron complex.